Optimized crispr-cas double nickase systems, methods and compositions for sequence manipulation

ABSTRACT

The invention provides for delivery, engineering and optimization of systems, methods, and compositions for manipulation of sequences and/or activities of target sequences. Provided are vectors and vector systems, some of which encode one or more components of a CRISPR complex, as well as methods for the design and use of such vectors. Also provided are methods of directing CRISPR complex formation in prokaryotic and eukaryotic cells to ensure enhanced specificity for target recognition and avoidance of toxicity.

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application is a Continuation-In-Part of International ApplicationNumber PCT/US2014/041803 filed on Jun. 10, 2014 which published as PCTPublication Number WO2014/204725 on Dec. 12, 2014. Priority is claimedfrom U.S. provisional patent applications 61/836,123, filed Jun. 17,2013, 61/847,537, filed Jul. 17, 2013, 61/862,355, filed Aug. 5, 2013,61/871,301, filed Aug. 28, 2013, 61/915,383, filed Dec. 12, 2013, andPCT/US2013/074667, filed Dec. 12, 2013, as to which for purposes of theUnited States, this application is also a continuation-in-part.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant no. MH100706awarded by the National Institutes of Health. The government has certainrights in the invention.

The foregoing applications, and all documents cited therein or duringtheir prosecution (“appin cited documents”) and all documents cited orreferenced in the appin cited documents, and all documents cited orreferenced herein (“herein cited documents”), and all documents cited orreferenced in herein cited documents, together with any manufacturer'sinstructions, descriptions, product specifications, and product sheetsfor any products mentioned herein or in any document incorporated byreference herein, are hereby incorporated herein by reference, and maybe employed in the practice of the invention. More specifically, allreferenced documents are incorporated by reference to the same extent asif each individual document was specifically and individually indicatedto be incorporated by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 14, 2015, isnamed 44790172022 SLtxt and is 253,354 bytes in size.

FIELD OF THE INVENTION

The present invention generally relates to the delivery, engineering andoptimization of systems, methods and compositions used for the controlof gene expression involving sequence targeting, such as genomeperturbation or gene-editing, that relate to Clustered RegularlyInterspaced Short Palindromic Repeats (CRISPR) and components thereof.

BACKGROUND OF THE INVENTION

Recent advances in genome sequencing techniques and analysis methodshave significantly accelerated the ability to catalog and map geneticfactors associated with a diverse range of biological functions anddiseases. Precise genome targeting technologies are needed to enablesystematic reverse engineering of causal genetic variations by allowingselective perturbation of individual genetic elements, as well as toadvance synthetic biology, biotechnological, and medical applications.Although genome-editing techniques such as designer zinc fingers,transcription activator-like effectors (TALEs), or homing meganucleasesare available for producing targeted genome perturbations, there remainsa need for new genome engineering technologies that are affordable, easyto set up, scalable, and amenable to targeting multiple positions withinthe eukaryotic genome.

SUMMARY OF THE INVENTION

The CRISPR-Cas system does not require the generation of customizedproteins to target specific sequences but rather a single Cas enzyme canbe programmed by a short RNA molecule to recognize a specific DNAtarget. Adding the CRISPR-Cas system to the repertoire of genomesequencing techniques and analysis methods may significantly simplifythe methodology and accelerate the ability to catalog and map geneticfactors associated with a diverse range of biological functions anddiseases. To utilize the CRISPR-Cas system effectively for genomeediting without deleterious effects, it is critical to understandaspects of engineering and optimization of these genome engineeringtools, which are aspects of the claimed invention.

There exists a pressing need for alternative and robust systems andtechniques for sequence targeting with a wide array of applications.Aspects of this invention address this need and provides relatedadvantages. An exemplary CRISPR complex comprises a CRISPR enzymecomplexed with a guide sequence hybridized to a target sequence withinthe target polynucleotide. The guide sequence is linked to a tracr matesequence, which in turn hybridizes to a tracr sequence.

In one aspect, the invention provides methods for using one or moreelements of a CRISPR system. The CRISPR complex of the inventionprovides an effective means for modifying a target polynucleotide. TheCRISPR complex of the invention has a wide variety of utilitiesincluding modifying (e.g., deleting, inserting, translocating,inactivating, activating) a target polynucleotide in a multiplicity ofcell types. In one aspect, the cell is a eukaryotic cell. In one aspect,the cell is a prokaryotic cell. As such the CRISPR complex of theinvention has a broad spectrum of applications in, e.g., gene or genomeediting, gene therapy, drug discovery, drug screening, diseasediagnosis, and prognosis. An exemplary CRISPR complex comprises a CRISPRenzyme complexed with a guide sequence hybridized to a target sequencewithin the target polynucleotide. In one aspect, the guide sequence islinked to a tracr mate sequence, which in turn hybridizes to a tracrsequence. In one aspect, the CRISPR enzyme is a nickase.

Aspects of the invention relate to Cas9 enzymes having improved targetspecificity in a CRISPR-Cas9 system, having guide RNAs with optimalactivity, with Cas9 enzymes that are smaller in length than wild-typeCas9 enzymes (and nucleic acid molecules coding therefor), and chimericCas9 enzymes, as well as methods of improving the target specificity ofa Cas9 enzyme or of designing a CRISPR-Cas9 system comprising designingor preparing guide RNAs having optimal activity and/or selecting orpreparing a Cas9 enzyme having a smaller size or length than wild-typeCas9 whereby packaging a nucleic acid coding such construct into adelivery vector is advantageous as there is less coding therefor in thedelivery vector than for wild-type Cas9, and/or generating chimeric Cas9enzymes.

Also provided are uses of the present sequences, vectors, enzymes orsystems, in medicine. Also provided are uses of the same in gene orgenome editing.

In an additional aspect of the invention, a Cas9 enzyme may comprise oneor more mutations and may be used as a generic DNA binding protein withor without fusion to a functional domain. The mutations may beartificially introduced mutations or gain- or loss-of-functionmutations. The mutations may include but are not limited to mutations inone of the catalytic domains (e.g., at residues D10 and H840). Furthermutations have been characterized and may be used in the inventiveconstructs. In one aspect of the invention, the mutated Cas9 enzyme maybe fused to a protein domain, e.g., such as a transcriptional activationdomain. In one aspect, the transcriptional activation domain may beVP64. Other aspects of the invention relate to the mutated Cas 9 enzymebeing fused to domains which include but are not limited to atranscriptional repressor, a recombinase, a transposase, a histoneremodeler, a DNA methyltransferase, a cryptochrome, a lightinducible/controllable domain or a chemically inducible/controllabledomain.

In a further embodiment, the invention provides for methods to generatemutant tracrRNA and direct repeat sequences or mutant chimeric guidesequences that allow for enhancing performance of these RNAs in cells.Aspects of the invention also provide for selection of said sequences.

Aspects of the invention also provide for methods of simplifying thecloning and delivery of components of the CRISPR complex. In thepreferred embodiment of the invention, a suitable promoter, such as theU6 promoter, is amplified with a DNA oligo and positioned contiguous toand upstream of a sequence encoding the guide RNA. The resulting PCRproduct can then be transfected into cells to drive expression of theguide RNA. Aspects of the invention also relate to the guide RNA beingtranscribed in vitro or ordered from a synthesis company and directlytransfected.

In one aspect, the invention provides for methods to improve activity byusing a more active polymerase. In one aspect, a T7 promoter may beinserted contiguous to and upstream of a sequence encoding a guide RNA.In a preferred embodiment, the expression of guide RNAs under thecontrol of the T7 promoter is driven by the expression of the T7polymerase in the cell. In an advantageous embodiment, the cell is aeukaryotic cell. In a preferred embodiment the eukaryotic cell is ahuman cell. In a more preferred embodiment the human cell is a patientspecific cell.

In one aspect, the invention provides for methods of reducing thetoxicity of Cas enzymes. In certain aspects, the Cas enzyme is any Cas9as described herein, for instance any naturally-occurring bacterial Cas9as well as any chimaeras, mutants, homologs or orthologs. In one aspect,the Cas enzyme is a nickase. In one embodiment, the Cas9 is deliveredinto the cell in the form of mRNA. This allows for the transientexpression of the enzyme thereby reducing toxicity. In anotherembodiment, the Cas9 is delivered into the cell in the nucleotideconstruct that encodes and expresses the Cas9 enzyme. In anotherembodiment, the invention also provides for methods of expressing Cas9under the control of an inducible promoter the constructs used therein.

In another aspect, the invention provides for methods of improving thein vivo applications of the CRISPR-Cas system. In the preferredembodiment, the Cas enzyme is wildtype Cas9 or any of the modifiedversions described herein, including any naturally-occurring bacterialCas9 as well as any chimaeras, mutants, homologs or orthologs. In oneaspect, the Cas enzyme is a nickase. An advantageous aspect of theinvention provides for the selection of Cas9 homologs that are easilypackaged into viral vectors for delivery. Cas9 orthologs typically sharethe general organization of 3-4 RuvC domains and a HNH domain. The 5′most RuvC domain cleaves the non-complementary strand, and the HNHdomain cleaves the complementary strand. All notations are in referenceto the guide sequence.

The catalytic residue in the 5′ RuvC domain is identified throughhomology comparison of the Cas9 of interest with other Cas9 orthologs(from S. pyogenes type II CRISPR locus, S. thermophilus CRISPR locus 1,S. thermophilus CRISPR locus 3, and Franciscilla novicida type II CRISPRlocus), and the conserved Asp residue is mutated to alanine to convertCas9 into a complementary-strand nicking enzyme. Similarly, theconserved His and Asn residues in the HNH domains are mutated to Alanineto convert Cas9 into a non-complementary-strand nicking enzyme.

In some embodiments, the CRISPR enzyme is a type I or III CRISPR enzyme,preferably a type II CRISPR enzyme. This type II CRISPR enzyme may beany Cas enzyme. A Cas enzyme may be identified Cas9 as this can refer tothe general class of enzymes that share homology to the biggest nucleasewith multiple nuclease domains from the type II CRISPR system. Mostpreferably, the Cas9 enzyme is from, or is derived from, spCas9 orsaCas9. By derived, Applicants mean that the derived enzyme is largelybased, in the sense of having a high degree of sequence homology with, awildtype enzyme, but that it has been mutated (modified) in some way asdescribed herein

It will be appreciated that the terms Cas and CRISPR enzyme aregenerally used herein interchangeably, unless otherwise apparent. Asmentioned above, many of the residue numberings used herein refer to theCas9 enzyme from the type II CRISPR locus in Streptococcus pyogenes(annotated alternatively as SpCas9 or spCas9). However, it will beappreciated that this invention includes many more Cas9s from otherspecies of microbes, such as SpCas9 derived from S. pyogenes, SaCas9derived from S. aureus, StlCas9 derived from S. thermophilus and soforth. Further examples are provided herein.

An example of a codon optimized sequence, in this instance optimized forhumans (i.e. being optimized for expression in humans) is providedherein, see the SaCas9 human codon optimized sequence. Whilst this ispreferred, it will be appreciated that other examples are possible andcodon optimization for a host species other than human, or for codonoptimization for specific organs such as the brain, is known.

In further embodiments, the invention provides for methods of enhancingthe function of Cas9 by generating chimeric Cas9 proteins. These methodsmay comprise fusing N-terminal fragments of one Cas9 homolog withC-terminal fragments of another Cas9 homolog. These methods also allowfor the selection of new properties displayed by the chimeric proteins.

It will be appreciated that in the present methods, where the organismis an animal or a plant, the modification may occur ex vivo or in vitro,for instance in a cell culture and in some instances not in vivo. Inother embodiments, it may occur in vivo.

In one aspect, the invention provides a method of modifying an organismor a non-human organism by manipulation of a target sequence in agenomic locus of interest comprising: delivering a non-naturallyoccurring or engineered composition comprising:

A)—I. a CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence,wherein the polynucleotide sequence comprises:

-   -   (a) a guide sequence capable of hybridizing to a target sequence        in a eukaryotic cell,    -   (b) a tracr mate sequence, and    -   (c) a tracr sequence, and        II. a polynucleotide sequence encoding a CRISPR enzyme        comprising at least one or more nuclear localization sequences,        wherein (a), (b) and (c) are arranged in a 5′ to 3′ orientation,        wherein when transcribed, the tracr mate sequence hybridizes to        the tracr sequence and the guide sequence directs        sequence-specific binding of a CRISPR complex to the target        sequence, and        wherein the CRISPR complex comprises the CRISPR enzyme complexed        with (1) the guide sequence that is hybridizable to the target        sequence, and (2) the tracr mate sequence that is hybridized to        the tracr sequence and the polynucleotide sequence encoding a        CRISPR enzyme is DNA or RNA,        or        (B) I. polynucleotides comprising:    -   (a) a guide sequence capable of hybridizing to a target sequence        in a prokaryotic cell, and    -   (b) at least one or more tracr mate sequences,        II. a polynucleotide sequence encoding a CRISPR enzyme, and        III. a polynucleotide sequence comprising a tracr sequence,        wherein when transcribed, the tracr mate sequence hybridizes to        the tracr sequence and the guide sequence directs        sequence-specific binding of a CRISPR complex to the target        sequence, and        wherein the CRISPR complex comprises the CRISPR enzyme complexed        with (1) the guide sequence that is hybridizable to the target        sequence, and (2) the tracr mate sequence that is hybridized to        the tracr sequence, and the polynucleotide sequence encoding a        CRISPR enzyme is DNA or RNA.

Any or all of the polynucleotide sequence encoding a CRISPR enzyme,guide sequence, tracr mate sequence or tracr sequence may be RNA, DNA ora combination of RNA and DNA. In one aspect, the polynucleotidescomprising the sequence encoding a CRISPR enzyme, the guide sequence,tracr mate sequence or tracr sequence are RNA. In one aspect, thepolynucleotides comprising the sequence encoding a CRISPR enzyme, theguide sequence, tracr mate sequence or tracr sequence are DNA. In oneaspect, the polynucleotides are a mixture of DNA and RNA, wherein someof the polynucleotides comprising the sequence encoding one or more ofthe CRISPR enzyme, the guide sequence, tracr mate sequence or tracrsequence are DNA and some of the polynucleotides are RNA. In one aspect,the polynucleotide comprising the sequence encoding the CRISPR enzyme isa DNA and the guide sequence, tracr mate sequence or tracr sequence areRNA. The one or more polynucleotides comprising the sequence encoding aCRISPR enzyme, the guide sequence, tracr mate sequence or tracr sequencemay be delivered via nanoparticles, exosomes, microvesicles, or agene-gun.

It will be appreciated that where reference is made to a polynucleotide,where that polynucleotide is RNA and is said to ‘comprise’ a featuresuch as a tracr mate sequence, the RNA sequence includes the feature.Where the polynucleotide is DNA and is said to comprise a feature such atracr mate sequence, the DNA sequence is or can be transcribed into theRNA that comprises the feature at issue. Where the feature is a protein,such as the CRISPR enzyme, the DNA or RNA sequence referred to is, orcan be, translated (and in the case of DNA transcribed first).Furthermore, in cases where an RNA encoding the CRISPR enzyme isprovided to a cell, it is understood that the RNA is capable of beingtranslated by the cell into which it is delivered.

Accordingly, in certain embodiments the invention provides a method ofmodifying an organism, including a prokaryotic organism or a eukaryoticorganism such as a plant or an animal, e.g., a mammal including human ora non-human mammal or organism, by manipulation of a target sequence ina genomic locus of interest comprising delivering a non-naturallyoccurring or engineered composition comprising a viral or plasmid vectorsystem comprising one or more viral or plasmid vectors operably encodinga composition for expression thereof, wherein the composition comprises:

(A) a non-naturally occurring or engineered composition comprising avector system comprising one or more vectors comprising I. a firstregulatory element operably linked to a CRISPR-Cas system chimeric RNA(chiRNA) polynucleotide sequence, wherein the polynucleotide sequencecomprises (a) a guide sequence capable of hybridizing to a targetsequence in a eukaryotic cell, (b) a tracr mate sequence, and (c) atracr sequence, and II. a second regulatory element operably linked toan enzyme-coding sequence encoding a CRISPR enzyme comprising zero or atleast one or more nuclear localization sequences (or optionally at leastone or more nuclear localization sequences as some embodiments caninvolve no NLS), wherein (a), (b) and (c) are arranged in a 5′ to 3′orientation, wherein components I and II are located on the same ordifferent vectors of the system, wherein when transcribed, the tracrmate sequence hybridizes to the tracr sequence and the guide sequencedirects sequence-specific binding of a CRISPR complex to the targetsequence, and wherein the hybridizable complex comprises the CRISPRenzyme complexed with (1) the guide sequence that is hybridizable to thetarget sequence, and (2) the tracr mate sequence that is hybridized tothe tracr sequence, or

(B) a non-naturally occurring or engineered composition comprising avector system comprising one or more vectors comprising I. a firstregulatory element operably linked to (a) a guide sequence capable ofhybridizing to a target sequence in a prokaryotic cell, and (b) at leastone or more tracr mate sequences, II. a second regulatory elementoperably linked to an enzyme-coding sequence encoding a CRISPR enzyme,and III. a third regulatory element operably linked to a tracr sequence,wherein components I, II and III are located on the same or differentvectors of the system, wherein when transcribed, the tracr mate sequencehybridizes to the tracr sequence and the guide sequence directssequence-specific binding of a CRISPR complex to the target sequence,and wherein the CRISPR complex comprises the CRISPR enzyme complexedwith (1) the guide sequence that is hybridizable to the target sequence,and (2) the tracr mate sequence that is hybridized to the tracrsequence. In one aspect, the CRISPR enzyme comprises one or moremutations in one of the catalytic domains. In one aspct, the CRISPRenzyme is a nickase.

Preferably, the vector is a viral vector, such as a lenti- or baculo- orpreferably adeno-viral/adeno-associated viral vectors, but other meansof delivery are known (such as yeast systems, microvesicles, geneguns/means of attaching vectors to gold nanoparticles) and are provided.In some embodiments, one or more of the viral or plasmid vectors may bedelivered via nanoparticles, exosomes, microvesicles, or a gene-gun.

By manipulation of a target sequence, Applicants also mean theepigenetic manipulation of a target sequence. This may be of thechromatin state of a target sequence, such as by modification of themethylation state of the target sequence (i.e. addition or removal ofmethylation or methylation patterns or CpG islands), histonemodification, increasing or reducing accessibility to the targetsequence, or by promoting or reducing 3D folding.

It will be appreciated that where reference is made to a method ofmodifying an organism, including a prokaryotic organism or a eukaryoticorganism such as a plant or an animal, e.g., a mammal including human ora non-human mammal or organism) by manipulation of a target sequence ina genomic locus of interest, this may apply to the organism (or mammal)as a whole or just a single cell or population of cells from thatorganism (if the organism is multicellular). In the case of humans, forinstance, Applicants envisage, inter alfa, a single cell or a populationof cells and these may preferably be modified ex vivo and thenre-introduced. In this case, a biopsy or other tissue or biologicalfluid sample may be necessary. Stem cells are also particularlypreferred in this regard. But, of course, in vivo embodiments are alsoenvisaged.

In certain embodiments the invention provides a method of treating orinhibiting a condition caused by a defect in a target sequence in agenomic locus of interest in a subject (e.g., mammal or human) or anon-human subject (e.g., mammal) in need thereof comprising modifyingthe human subject or a non-human subject by manipulation of the targetsequence and wherein the condition is susceptible to treatment orinhibition by manipulation of the target sequence comprising providingtreatment comprising: delivering a non-naturally occurring or engineeredcomposition comprising an AAV vector system comprising one or more AAVvectors comprising operably encoding a composition for expressionthereof, wherein the target sequence is manipulated by the compositionwhen expressed, wherein the composition comprises:

(A) a non-naturally occurring or engineered composition comprising avector system comprising one or more vectors comprising I. a firstregulatory element operably linked to a CRISPR-Cas system chimeric RNA(chiRNA) polynucleotide sequence, wherein the polynucleotide sequencecomprises (a) a guide sequence capable of hybridizing to a targetsequence in a eukaryotic cell, (b) a tracr mate sequence, and (c) atracr sequence, and II. a second regulatory element operably linked toan enzyme-coding sequence encoding a CRISPR enzyme comprising zero or atleast one or more nuclear localization sequences (or optionally at leastone or more nuclear localization sequences as some embodiments caninvolve no NLS) wherein (a), (b) and (c) are arranged in a 5′ to 3′orientation, wherein components I and II are located on the same ordifferent vectors of the system, wherein when transcribed, the tracrmate sequence hybridizes to the tracr sequence and the guide sequencedirects sequence-specific binding of a CRISPR complex to the targetsequence, and wherein the CRISPR complex comprises the CRISPR enzymecomplexed with (1) the guide sequence that is hybridizable to the targetsequence, and (2) the tracr mate sequence that is hybridized to thetracr sequence, or

(B) a non-naturally occurring or engineered composition comprising avector system comprising one or more vectors comprising I. a firstregulatory element operably linked to (a) a guide sequence capable ofhybridizing to a target sequence in a prokaryotic cell, and (b) at leastone or more tracr mate sequences, II. a second regulatory elementoperably linked to an enzyme-coding sequence encoding a CRISPR enzyme,and III. a third regulatory element operably linked to a tracr sequence,wherein components I, II and III are located on the same or differentvectors of the system, wherein when transcribed, the tracr mate sequencehybridizes to the tracr sequence and the guide sequence directssequence-specific binding of a CRISPR complex to the target sequence,and wherein the CRISPR complex comprises the CRISPR enzyme complexedwith (1) the guide sequence that is hybridizable to the target sequence,and (2) the tracr mate sequence that is hybridized to the tracrsequence.

Some methods of the invention can include inducing expression. In somemethods of the invention the organism or subject is a eukaryote,including e.g., a plant or an animal (including mammal, including human)or a non-human eukaryote or a non-human animal or a non-human mammal. Insome methods of the invention the organism or subject is a plant. Insome methods of the invention the organism or subject is a mammal or anon-human mammal. In some methods of the invention the organism orsubject is algae. In some methods of the invention the viral vector isan AAV. In some methods of the invention the viral vector is alentivirus-derived vector. In some methods of the invention the viralvector is an Agrobacterium Ti or Ri plasmid for use in plants. In somemethods of the invention the CRISPR enzyme is a Cas9. In some methods ofthe invention the CRISPR enzyme copmprises one or more mutations in oneof the catalytic domains. In some methods of the invention the CRISPRenzyme is a Cas9 nickase. In some methods of the invention theexpression of the guide sequence is under the control of a T7 promoterthat is driven by the expression of T7 polymerase. In some methods ofthe invention the expression of the guide sequence is under the controlof a U6 promoter.

By manipulation of a target sequence, Applicants mean the alteration ofthe target sequence, which may include the epigenetic manipulation of atarget sequence. This epigenetic manipulation may be of the chromatinstate of a target sequence, such as by modification of the methylationstate of the target sequence (i.e., addition or removal of methylationor methylation patterns or CpG islands), histone modification,increasing or reducing accessibility to the target sequence, or bypromoting or reducing 3D folding.

It will be appreciated that where reference is made to a method ofmodifying an organism or a non-human organism by manipulation of atarget sequence in a genomic locus of interest, this may apply to theorganism as a whole or just a single cell or population of cells fromthat organism (if the organism is multicellular). In the case of humans,for instance, Applicants envisage, inter alfa, a single cell or apopulation of cells and these may preferably be modified ex vivo andthen re-introduced. In this case, a biopsy or other tissue or biologicalfluid sample may be necessary. Stem cells are also particularlypreferred in this regard. But, of course, in vivo embodiments are alsoenvisaged.

In certain embodiments the invention provides a method of treating orinhibiting a condition caused by a defect in a target sequence in agenomic locus of interest in a subject or a non-human subject in needthereof comprising modifying the subject or a non-human subject bymanipulation of the target sequence and wherein the condition issusceptible to treatment or inhibition by manipulation of the targetsequence comprising providing treatment comprising: delivering anon-naturally occurring or engineered composition comprising a vectorsystem comprising one or more vectors comprising operably encoding acomposition for expression thereof, wherein the target sequence ismanipulated by the composition when expressed, wherein the compositioncomprises:

(A) a non-naturally occurring or engineered composition comprising avector system comprising one or more vectors comprising I. a firstregulatory element operably linked to a CRISPR-Cas system chimeric RNA(chiRNA) polynucleotide sequence, wherein the chiRNA polynucleotidesequence comprises (a) a guide sequence capable of hybridizing to atarget sequence in a eukaryotic cell, (b) a tracr mate sequence, and (c)a tracr sequence, and II. a second regulatory element operably linked toan enzyme-coding sequence encoding a CRISPR enzyme comprising zero or atleast one or more nuclear localization sequences (or optionally at leastone or more nuclear localization sequences as some embodiments caninvolve no NLS) wherein (a), (b) and (c) are arranged in a 5′ to 3′orientation, wherein components I and II are located on the same ordifferent vectors of the system, wherein when transcribed, the tracrmate sequence hybridizes to the tracr sequence and the guide sequencedirects sequence-specific binding of a CRISPR complex to the targetsequence, and wherein the CRISPR complex comprises the CRISPR enzymecomplexed with (1) the guide sequence that is hybridizable to the targetsequence, and (2) the tracr mate sequence that is hybridized to thetracr sequence, or

(B) a non-naturally occurring or engineered composition comprising avector system comprising one or more vectors comprising I. a firstregulatory element operably linked to (a) a guide sequence capable ofhybridizing to a target sequence in a prokaryotic cell, and (b) at leastone or more tracr mate sequences, II. a second regulatory elementoperably linked to an enzyme-coding sequence encoding a CRISPR enzyme,and III. a third regulatory element operably linked to a tracr sequence,wherein components I, II and III are located on the same or differentvectors of the system, wherein when transcribed, the tracr mate sequencehybridizes to the tracr sequence and the guide sequence directssequence-specific binding of a CRISPR complex to the target sequence,and wherein the CRISPR complex comprises the CRISPR enzyme complexedwith (1) the guide sequence that is hybridizable to the target sequence,and (2) the tracr mate sequence that is hybridized to the tracrsequence. In an embodiment for use in a eukaryotic cell, the vectorsystem comprises a viral vector system, e.g., an AAV vector or AAVvector system or a lentivirus-derived vector system or a tobacco mosaicvirus-derived system.

Some methods of the invention can include inducing expression. In somemethods of the invention the organism or subject is a eukaryote,including e.g., a plant or an animal (including mammal, including human)or a non-human eukaryote or a non-human animal. In some methods of theinvention the organism or subject is a plant. In some methods of theinvention the organism or subject is a mammal or a non-human mammal. Insome methods of the invention the organism or subject is algae. In somemethods of the invention the viral vector is an AAV. In some methods ofthe invention the viral vector is a lentiviral vector. In some methodsof the invention the viral vector is a tobacco mosaic virus vector. Insome methods of the invention the CRISPR enzyme is a Cas9. In somemethods of the invention the CRISPR enzyme copmprises one or moremutations in one of the catalytic domains. In some methods of theinvention the CRISPR enzyme is a Cas9 nickase. In some methods of theinvention the expression of the guide sequence is under the control ofthe T7 promoter is driven by the expression of T7 polymerase. In somemethods of the invention the expression of the guide sequence is underthe control of a U6 promoter.

The invention in some embodiments comprehends a method of delivering aCRISPR enzyme comprising delivering to a cell mRNA encoding the CRISPRenzyme. In some of these methods the CRISPR enzyme is a Cas9.

The invention in some embodiments comprehends a method of preparing theAAV vector of the invention comprising transfecting one or moreplasmid(s) containing or consisting essentially of nucleic acidmolecule(s) coding for the AAV into AAV-infectable cells, and supplyingAAV rep and/or cap obligatory for replication and packaging of the AAV.In some embodiments the AAV rep and/or cap obligatory for replicationand packaging of the AAV are supplied by transfecting the cells withhelper plasmid(s) or helper virus(es). In some embodiments the helpervirus is a poxvirus, adenovirus, herpesvirus or baculovirus. In someembodiments the poxvirus is a vaccinia virus. In some embodiments thecells are mammalian cells. And in some embodiments the cells are insectcells and the helper virus is baculovirus.

In plants, pathogens are often host-specific. For example, Fusariumoxysporum f sp. lycopersici causes tomato wilt but attacks only tomato,and F. oxysporum f. dianthii Puccinia graminis f sp. tritici attacksonly wheat. Plants have existing and induced defenses to resist mostpathogens. Mutations and recombination events across plant generationslead to genetic variability that gives rise to susceptibility,especially as pathogens reproduce with more frequency than plants. Inplants there can be non-host resistance, e.g., the host and pathogen areincompatible. There can also be Horizontal Resistance, e.g., partialresistance against all races of a pathogen, typically controlled by manygenes and Vertical Resistance, e.g., complete resistance to some racesof a pathogen but not to other races, typically controlled by a fewgenes. In a Gene-for-Gene level, plants and pathogens evolve together,and the genetic changes in one balance changes in other. Accordingly,using Natural Variability, breeders combine most useful genes for Yield,Quality, Uniformity, Hardiness, Resistance. The sources of resistancegenes include native or foreign Varieties, Heirloom Varieties, WildPlant Relatives, and Induced Mutations, e.g., treating plant materialwith mutagenic agents. Using the present invention, plant breeders areprovided with a new tool to induce mutations. Accordingly, one skilledin the art can analyze the genome of sources of resistance genes, and inVarieties having desired characteristics or traits employ the presentinvention to induce the rise of resistance genes, with more precisionthan previous mutagenic agents and hence accelerate and improve plantbreeding programs.

The invention further comprehends a composition of the invention or aCRISPR enzyme thereof (including or alternatively mRNA encoding theCRISPR enzyme) for use in medicine. In some embodiments the inventioncomprehends a composition according to the invention or a CRISPR enzymethereof (including or alternatively mRNA encoding the CRISPR enzyme) foruse in a method according to the invention. In some embodiments theinvention provides for the use of a composition of the invention or aCRISPR enzyme thereof (including or alternatively mRNA encoding theCRISPR enzyme) in ex vivo gene or genome editing. In certain embodimentsthe invention comprehends use of a composition of the invention or aCRISPR enzyme thereof (including or alternatively mRNA encoding theCRISPR enzyme) in the manufacture of a medicament for ex vivo gene orgenome editing or for use in a method according of the invention. Insome methods of the invention the CRISPR enzyme copmprises one or moremutations in one of the catalytic domains. In some methods of theinvention the CRISPR enzyme is a Cas9 nickase. The invention comprehendsin some embodiments a composition of the invention or a CRISPR enzymethereof (including or alternatively mRNA encoding the CRISPR enzyme),wherein the target sequence is flanked at its 3′ end by a 5′-motiftermed a proto-spacer adjacent motif (PAM), especially where the Cas9 is(or is derived from) S. pyogenes or S. aureus Cas9. For example, asuitable PAM is 5′-NRG or 5′-NNGRR (where N is any Nucleotide) forSpCas9 or SaCas9 enzymes (or derived enzymes), respectively, asmentioned below.

It will be appreciated that SpCas9 or SaCas9 are those from or derivedfrom S. pyogenes or S. aureus Cas9.

Apects of the invention comprehend improving the specificity of a CRISPRenzyme, e.g. Cas9, mediated gene targeting and reducing the likelihoodof off-target modification by the CRISPR enzyme, e.g. Cas9. Theinvention in some embodiments comprehends a method of modifying anorganism or a non-human organism by minimizing off-target modificationsby manipulation of a first and a second target sequence on oppositestrands of a DNA duplex in a genomic locus of interest in a cell, saidmethod comprising delivering a non-naturally occurring or engineeredcomposition comprising:

I. a first CRISPR-Cas system chimeric RNA (chiRNA) polynucleotidesequence, wherein the first polynucleotide sequence comprises:

-   -   (a) a first guide sequence capable of hybridizing to the first        target sequence,    -   (b) a first tracr mate sequence, and    -   (c) a first tracr sequence,        II. a second CRISPR-Cas system chiRNA polynucleotide sequence,        wherein the second polynucleotide sequence comprises:    -   (a) a second guide sequence capable of hybridizing to the second        target sequence,    -   (b) a second tracr mate sequence, and    -   (c) a second tracr sequence, and        III. a polynucleotide sequence encoding a CRISPR enzyme        comprising zero or at least one or more nuclear localization        sequences and comprising one or more mutations in the CRISPR        enzyme,        wherein (a), (b) and (c) of I. and II. are arranged in a 5′ to        3′ orientation, wherein when transcribed, the first and the        second tracr mate sequence hybridize to the first and second        tracr sequence respectively and the first and the second guide        sequence directs sequence-specific binding of a first and a        second CRISPR complex to the first and second target sequences        respectively, wherein the first CRISPR complex comprises the        CRISPR enzyme complexed with (1) the first guide sequence that        is hybridizable to the first target sequence, and (2) the first        tracr mate sequence that is hybridized to the first tracr        sequence, wherein the second CRISPR complex comprises the CRISPR        enzyme complexed with (1) the second guide sequence that is        hybridizable to the second target sequence, and (2) the second        tracr mate sequence that is hybridized to the second tracr        sequence,        wherein the polynucleotide sequence encoding a CRISPR enzyme is        DNA or RNA, and wherein the first guide sequence directs        cleavage of one strand of the DNA duplex near the first target        sequence and the second guide sequence directs cleavage of        opposite strand of the DNA duplex near the second target        sequence, thereby inducing a break in the DNA, thereby modifying        the organism or the non-human organism by minimizing off-target        modifications. In one aspect, the first nick and the second nick        in the DNA is offset relative to each other by at least one base        pair of the duplex. In one aspect, the first nick and the second        nick are offset relative to each other so that the resulting DNA        break has a 3′ overhang. In one aspct, the first nick and the        second nick are offset relative to each other so that the        resulting DNA break has a 5′ overhang. In one aspect, the first        nick and the second nick are positioned relative to each other        such that the overhang is at least 1 nt, at least 10 nt, at        least 15 nt, at least 26 nt, at least 30 nt, at least 50 nt or        more that at least 50 nt. Additional aspects of the invention        comprising the resulting offset double nicked DNA strand can be        appreciated by one skilled in the art, and exemplary uses of the        double nick system are provided herein.

In some methods of the invention any or all of the polynucleotidesequences encoding the CRISPR enzyme, the first and the second guidesequence, the first and the second tracr mate sequence or the first andthe second tracr sequence, is/are RNA. In further embodiments of theinvention the polynucleotides comprising the sequence encoding theCRISPR enzyme, the first and the second guide sequence, the first andthe second tracr mate sequence or the first and the second tracrsequence, is/are RNA and are delivered via nanoparticles, exosomes,microvesicles, or a gene-gun. In certain embodiments of the invention,the first and second tracr mate sequence share 100% identity and/or thefirst and second tracr sequence share 100% identity. In preferredembodiments of the invention the CRISPR enzyme is a Cas9 enzyme, e.g.SpCas9. In an aspect of the invention the CRISPR enzyme comprises one ormore mutations in one of the catalytic domains, wherein the one or moremutations is selected from the group consisting of D10A, E762A, andD986A in the RuvC domain or the one or more mutations is selected fromthe group consisting of H840A, N854A and N863A in the HNH domain. In ahighly preferred embodiment the CRISPR enzyme has the D10A mutation orthe H840A mutation.

In preferred methods of the invention the first guide sequence directingcleavage of one strand of the DNA duplex near the first target sequenceand the second guide sequence directing cleavage of the opposite strandnear the second target sequence results in a 5′ overhang. In embodimentsof the invention the 5′ overhang is at most 200 base pairs, preferablyat most 100 base pairs, or more preferably at most 50 base pairs. Inembodiments of the invention the 5′ overhang is at least 26 base pairs,preferably at least 30 base pairs or more preferably 34-50 base pairs or1-34 base pairs. In other preferred methods of the invention the firstguide sequence directing cleavage of one strand of the DNA duplex nearthe first target sequence and the second guide sequence directingcleavage of other strand near the second target sequence results in ablunt cut or a 3′ overhang. In embodiments of the invention the 3′overhang is at most 150, 100 or 25 base pairs or at least 15, 10 or 1base pairs. In preferred embodiments the 3′ overhang is 1-100 basepairs.

The invention in some embodiments comprehends a method of modifying anorganism or a non-human organism by minimizing off-target modificationsby manipulation of a first and a second target sequence on oppositestrands of a DNA duplex in a genomic locus of interest in a cell, themethod comprising delivering a non-naturally occurring or engineeredcomposition comprising a vector system comprising one or more vectorscomprising

I. a first regulatory element operably linked to

-   -   (a) a first guide sequence capable of hybridizing to the first        target sequence, and    -   (b) at least one or more tracr mate sequences,        II. a second regulatory element operably linked to    -   (a) a second guide sequence capable of hybridizing to the second        target sequence, and    -   (b) at least one or more tracr mate sequences,        III. a third regulatory element operably linked to an        enzyme-coding sequence encoding a CRISPR enzyme, and        IV. a fourth regulatory element operably linked to a tracr        sequence,        wherein components I, II, III and IV are located on the same or        different vectors of the system, and        when transcribed, the tracr mate sequence hybridizes to the        tracr sequence and the first and the second guide sequence        directs sequence-specific binding of a first and a second CRISPR        complex to the first and second target sequences respectively,        wherein the first CRISPR complex comprises the CRISPR enzyme        complexed with (1) the first guide sequence that is hybridizable        to the first target sequence, and (2) the tracr mate sequence        that is hybridized to the tracr sequence, wherein the second        CRISPR complex comprises the CRISPR enzyme complexed with (1)        the second guide sequence that is hybridizable to the second        target sequence, and (2) the tracr mate sequence that is        hybridized to the tracr sequence, wherein the polynucleotide        sequence encoding a CRISPR enzyme is DNA or RNA, and wherein the        first guide sequence directs cleavage of one strand of the DNA        duplex near the first target sequence and the second guide        sequence directs cleavage of other strand near the second target        sequence inducing a double strand break, thereby modifying the        organism or the non-human organism by minimizing off-target        modifications.

In some methods of the invention any or all of the polynucleotidesequence encoding the CRISPR enzyme, the first and the second guidesequence, the first and the second tracr mate sequence or the first andthe second tracr sequence, is/are RNA. In further embodiments of theinvention the first and second tracr mate sequence share 100% identityand/or the first and second tracr sequence share 100% identity. Inpreferred embodiments of the invention the CRISPR enzyme is a Cas9enzyme, e.g. SpCas9. In an aspect of the invention the CRISPR enzymecomprises one or more mutations in one of the catalytic domains, whereinthe one or more mutations is selected from the group consisting of D10A,E762A, and D986A in the RuvC domain or the one or more mutations isselected from the group consisting of H840A, N854A and N863A in the HNHdomain. In a highly preferred embodiment the CRISPR enzyme has the D10Amutation or the H840A mutation. As CRISPR enzymes or Cas proteins maystill promote residual NHEJ activity with either the D10A or the H840Amutations, through out the application the invention comprehends thegeneration of even more precise nickases that further reduces NHEJactivity by introducing multiple mutations in the CRISPR enzyme or theCas protein. A better version of the D10A nicakse may include themutaions D10A, E762A and D986A (or some subset of these) and a betterversion of the H840A nickases may include the mutations H840A, N854A andN863A (or some subset of these).

In a further embodiment of the invention, one or more of the viralvectors are delivered via nanoparticles, exosomes, microvesicles, or agene-gun.

In preferred methods of the invention the first guide sequence directingcleavage of one strand of the DNA duplex near the first target sequenceand the second guide sequence directing cleavage of other strand nearthe second target sequence results in a 5′ overhang. In embodiments ofthe invention the 5′ overhang is at most 200 base pairs, preferably atmost 100 base pairs, or more preferably at most 50 base pairs. Inembodiments of the invention the 5′ overhang is at least 26 base pairs,preferably at least 30 base pairs or more preferably 34-50 base pairs or1-34 base pairs. In other preferred methods of the invention the firstguide sequence directing cleavage of one strand of the DNA duplex nearthe first target sequence and the second guide sequence directingcleavage of other strand near the second target sequence results in ablunt cut or a 3′ overhang. In embodiments of the invention the 3′overhang is at most 150, 100 or 25 base pairs or at least 15, 10 or 1base pairs. In preferred embodiments the 3′ overhang is 1-100 basepairs.

The invention in some embodiments comprehends a method of modifying agenomic locus of interest by minimizing off-target modifications byintroducing into a cell containing and expressing a double stranded DNAmolecule encoding the gene product an engineered, non-naturallyoccurring CRISPR-Cas system comprising a Cas protein having one or moremutations and two guide RNAs that target a first strand and a secondstrand of the DNA molecule respectively, whereby the guide RNAs targetthe DNA molecule encoding the gene product and the Cas protein nickseach of the first strand and the second strand of the DNA moleculeencoding the gene product, whereby expression of the gene product isaltered; and, wherein the Cas protein and the two guide RNAs do notnaturally occur together.

In preferred methods of the invention the Cas protein nicking each ofthe first strand and the second strand of the DNA molecule encoding thegene product results in a 5′ overhang. In embodiments of the inventionthe 5′ overhang is at most 200 base pairs, preferably at most 100 basepairs, or more preferably at most 50 base pairs. In embodiments of theinvention the 5′ overhang is at least 26 base pairs, preferably at least30 base pairs or more preferably 34-50 base pairs or 1-34 base pairs. Inother preferred methods of the invention the Cas protein nicking each ofthe first strand and the second strand of the DNA molecule encoding thegene product results in a 3′ overhang, wherein the 3′ overhang is 1-100base pairs.

Embodiments of the invention also comprehend the guide RNAs comprising aguide sequence fused to a tracr mate sequence and a tracr sequence. Inan aspect of the invention the Cas protein is codon optimized forexpression in a eukaryotic cell, wherein it may be a mammalian cell or ahuman cell. In further embodiments of the invention the Cas protein is atype II CRISPR-Cas protein, e.g. a Cas 9 protein. In a highly preferredembodiment the Cas protein is a Cas9 protein, e.g. SpCas9. In aspects ofthe invention the Cas protein has one or more mutations in one of thecatalytic domains, wherein the one or more mutations is selected fromthe group consisting of D10A, E762A, and D986A in the RuvC domain or theone or more mutations is selected from the group consisting of H840A,N854A and N863A in the HNH domain. In a highly preferred embodiment theCas protein has the D10A mutation or the H840A mutation.

Aspects of the invention relate to the expression of the gene productbeing decreased or a template polynucleotide being further introducedinto the DNA molecule encoding the gene product or an interveningsequence being excised precisely by allowing the two 5′ overhangs toreanneal and ligate or the activity or function of the gene productbeing altered or the expression of the gene product being increased. Inan embodiment of the invention, the gene product is a protein.

The invention also comprehends an engineered, non-naturally occurringCRISPR-Cas system comprising a Cas protein having one or more mutationsand two guide RNAs that target a first strand and a second strandrespectively of a double stranded DNA molecule encoding a gene productin a cell, whereby the guide RNAs target the DNA molecule encoding thegene product and the Cas protein nicks each of the first strand and thesecond strand of the DNA molecule encoding the gene product, wherebyexpression of the gene product is altered; and, wherein the Cas proteinand the two guide RNAs do not naturally occur together.

In aspects of the invention the guide RNAs may comprise a guide sequencefused to a tracr mate sequence and a tracr sequence. In an embodiment ofthe invention the Cas protein is a type II CRISPR-Cas protein. In anaspect of the invention the Cas protein is codon optimized forexpression in a eukaryotic cell, wherein it may be a mammalian cell or ahuman cell. In further embodiments of the invention the Cas protein is atype II CRISPR-Cas protein, e.g. a Cas 9 protein. In a highly preferredembodiment the Cas protein is a Cas9 protein, e.g. SpCas9. In aspects ofthe invention the Cas protein has one or more mutations in one of thecatalytic domains, wherein the one or more mutations is selected fromthe group consisting of D10A, E762A, and D986A in the RuvC domain or theone or more mutations is selected from the group consisting of H840A,N854A and N863A in the HNH domain. In a highly preferred embodiment theCas protein has the D10A mutation or the H840A mutation.

Aspects of the invention relate to the expression of the gene productbeing decreased or a template polynucleotide being further introducedinto the DNA molecule encoding the gene product or an interveningsequence being excised precisely by allowing the two 5′ or 3′ overhangsto reanneal and ligate or the activity or function of the gene productbeing altered or the expression of the gene product being increased. Inan embodiment of the invention, the gene product is a protein.

The invention also comprehends an engineered, non-naturally occurringvector system comprising one or more vectors comprising:

-   -   a) a first regulatory element operably linked to each of two        CRISPR-Cas system guide RNAs that target a first strand and a        second strand respectively of a double stranded DNA molecule        encoding a gene product,    -   b) a second regulatory element operably linked to a Cas protein,

wherein components (a) and (b) are located on same or different vectorsof the system, whereby the guide RNAs target the DNA molecule encodingthe gene product and the Cas protein nicks each of the first strand andthe second strand of the DNA molecule encoding the gene product, wherebyexpression of the gene product is altered; and, wherein the Cas proteinand the two guide RNAs do not naturally occur together.

In aspects of the invention the guide RNAs may comprise a guide sequencefused to a tracr mate sequence and a tracr sequence. In an embodiment ofthe invention the Cas protein is a type II CRISPR-Cas protein. In anaspect of the invention the Cas protein is codon optimized forexpression in a eukaryotic cell, wherein it may be a mammalian cell or ahuman cell. In further embodiments of the invention the Cas protein is atype II CRISPR-Cas protein, e.g. a Cas 9 protein. In a highly preferredembodiment the Cas protein is a Cas9 protein, e.g. SpCas9. In aspects ofthe invention the Cas protein has one or more mutations in one of thecatalytic domains, wherein the one or more mutations is selected fromthe group consisting of D10A, E762A, and D986A in the RuvC domain or theone or more mutations is selected from the group consisting of H840A,N854A and N863A in the HNH domain. In a highly preferred embodiment theCas protein has the D10A mutation or the H840A mutation.

Aspects of the invention relate to the expression of the gene productbeing decreased or a template polynucleotide being further introducedinto the DNA molecule encoding the gene product or an interveningsequence being excised precisely by allowing the two 5′ or 3′ overhangsto reanneal and ligate or the activity or function of the gene productbeing altered or the expression of the gene product being increased. Inan embodiment of the invention, the gene product is a protein. Inpreferred embodiments of the invention the vectors of the system areviral vectors. In a further embodiment, the vectors of the system aredelivered via nanoparticles, exosomes, microvesicles, or a gene-gun.

Aspects of the invention provide for methods of modifying an organismcomprising a first and a second target sequence on opposite strands of aDNA duplex in a genomic locus of interest in a cell by promotinghomology directed repair wherein the method may comprise delivering anon-naturally occurring or engineered composition comprising:

I. a first CRISPR-Cas system chimeric RNA (chiRNA) polynucleotidesequence, wherein the first polynucleotide sequence comprises:(a) a first guide sequence capable of hybridizing to the first targetsequence,(b) a first tracr mate sequence, and(c) a first tracr sequence,II. a second CRISPR-Cas system chiRNA polynucleotide sequence, whereinthe second polynucleotide sequence comprises:(a) a second guide sequence capable of hybridizing to the second targetsequence,(b) a second tracr mate sequence, and(c) a second tracr sequence, andIII. a polynucleotide sequence encoding a CRISPR enzyme comprising atleast one or more nuclear localization sequences and comprising one ormore mutations,IV. a repair template comprising a synthesized or engineeredsingle-stranded oligonucleotide, wherein (a), (b) and (c) are arrangedin a 5′ to 3′ orientation, wherein when transcribed, the first and thesecond tracr mate sequence hybridize to the first and second tracrsequence respectively and the first and the second guide sequencedirects sequence-specific binding of a first and a second CRISPR complexto the first and second target sequences respectively, wherein the firstCRISPR complex comprises the CRISPR enzyme complexed with (1) the firstguide sequence that is hybridizable to the first target sequence, and(2) the first tracr mate sequence that is hybridized to the first tracrsequence, wherein the second CRISPR complex comprises the CRISPR enzymecomplexed with (1) the second guide sequence that is hybridizable to thesecond target sequence, and (2) the second tracr mate sequence that ishybridized to the second tracr sequence, wherein the polynucleotidesequence encoding a CRISPR enzyme is DNA or RNA, wherein the first guidesequence directs cleavage of one strand of the DNA duplex near the firsttarget sequence and the second guide sequence directs cleavage of otherstrand near the second target sequence inducing a double strand break,and wherein the repair template is introduced into the DNA duplex byhomologous recombination, whereby the organism is modified.

In embodiments of the invention the repair template may further comprisea restriction endonuclease restriction site. In further embodiments, thefirst guide sequence directing cleavage of one strand of the DNA duplexnear the first target sequence and the second guide sequence directingcleavage of other strand near the second target sequence results in a 5′or 3′ overhang. In preferred embodiments of the invention, the 5′overhang is 1-200 base pairs or the 3′ overhang is 1-100 base pairs. Inother aspects, any or all of the polynucleotide sequence encoding theCRISPR enzyme, the first and the second guide sequence, the first andthe second tracr mate sequence or the first and the second tracrsequence, is/are RNA. In yet further aspects the polynucleotidescomprising the sequence encoding the CRISPR enzyme, the first and thesecond guide sequence, the first and the second tracr mate sequence orthe first and the second tracr sequence, is/are RNA and are deliveredvia nanoparticles, exosomes, microvesicles, or a gene-gun. In furtherembodiments of the invention the first and second tracr mate sequenceshare 100% identity and/or the first and second tracr sequence share100% identity. In preferred embodiments of the invention the CRISPRenzyme is a Cas9 enzyme, e.g. SpCas9. In an aspect of the invention theCRISPR enzyme comprises one or more mutations in one of the catalyticdomains, wherein the one or more mutations is selected from the groupconsisting of D10A, E762A, and D986A in the RuvC domain or the one ormore mutations is selected from the group consisting of H840A, N854A andN863A in the HNH domain. In a highly preferred embodiment the CRISPRenzyme has the D10A mutation or the H840A mutation.

Aspects of the invention also provide for methods of modifying anorganism comprising a first and a second target sequence on oppositestrands of a DNA duplex in a genomic locus of interest in a cell byfacilitating non homologous end joining (NHEJ) mediated ligationcomprising delivering a non-naturally occurring or engineeredcomposition comprising:

I. a first CRISPR-Cas system chimeric RNA (chiRNA) polynucleotidesequence, wherein the first polynucleotide sequence comprises:(a) a first guide sequence capable of hybridizing to the first targetsequence,(b) a first tracr mate sequence, and(c) a first tracr sequence,II. a second CRISPR-Cas system chiRNA polynucleotide sequence, whereinthe second polynucleotide sequence comprises:(a) a second guide sequence capable of hybridizing to the second targetsequence,(b) a second tracr mate sequence, and(c) a second tracr sequence, andIII. a polynucleotide sequence encoding a CRISPR enzyme comprising atleast one or more nuclear localization sequences and comprising one ormore mutations,IV. a repair template comprising a first set of overhangs,wherein (a), (b) and (c) are arranged in a 5′ to 3′ orientation, whereinwhen transcribed, the first and the second tracr mate sequence hybridizeto the first and second tracr sequence respectively and the first andthe second guide sequence directs sequence-specific binding of a firstand a second CRISPR complex to the first and second target sequencesrespectively, wherein the first CRISPR complex comprises the CRISPRenzyme complexed with (1) the first guide sequence that is hybridizableto the first target sequence, and (2) the first tracr mate sequence thatis hybridized to the first tracr sequence, wherein the second CRISPRcomplex comprises the CRISPR enzyme complexed with (1) the second guidesequence that is hybridizable to the second target sequence, and (2) thesecond tracr mate sequence that is hybridized to the second tracrsequence, wherein the polynucleotide sequence encoding a CRISPR enzymeis DNA or RNA, wherein the first guide sequence directs cleavage of onestrand of the DNA duplex near the first target sequence and the secondguide sequence directs cleavage of other strand near the second targetsequence inducing a double strand break with a second set of overhangs,wherein the first set of overhangs is compatible with and matches thesecond set of overhangs, and wherein the repair template is introducedinto the DNA duplex by ligation, whereby the organism is modified.

In some embodiments of the invention the repair template is asynthesized or engineered double-stranded oligonucleotide duplex or inother embodiments the repair template is generated from a piece of DNAthat is introduced into the cell and is enzymatically processed. Thisenzymatic processing may be carried out by endogenous enzymes or byenzymes (e.g. restriction endonucelases, nucleases or a pair ofnickases) that have been introduced into the cell so the compatibleoverhangs are generated on the repair template.

In further embodiments, the first guide sequence directing cleavage ofone strand of the DNA duplex near the first target sequence and thesecond guide sequence directing cleavage of other strand near the secondtarget sequence results in a 5′ or 3′ overhang. In preferred embodimentsof the invention, the 5′ overhang is 1-200 base pairs or the 3′ overhangis 1-100 base pairs. In other aspects, any or all of the polynucleotidesequence encoding the CRISPR enzyme, the first and the second guidesequence, the first and the second tracr mate sequence or the first andthe second tracr sequence, is/are RNA. In yet further aspects thepolynucleotides comprising the sequence encoding the CRISPR enzyme, thefirst and the second guide sequence, the first and the second tracr matesequence or the first and the second tracr sequence, is/are RNA and aredelivered via nanoparticles, exosomes, microvesicles, or a gene-gun. Infurther embodiments of the invention the first and second tracr matesequence share 100% identity and/or the first and second tracr sequenceshare 100% identity. In preferred embodiments of the invention theCRISPR enzyme is a Cas9 enzyme, e.g. SpCas9. In an aspect of theinvention the CRISPR enzyme comprises one or more mutations in one ofthe catalytic domains, wherein the one or more mutations is selectedfrom the group consisting of D10A, E762A, and D986A in the RuvC domainor the one or more mutations is selected from the group consisting ofH840A, N854A and N863A in the HNH domain. In a highly preferredembodiment the CRISPR enzyme has the D10A mutation or the H840Amutation.

In one aspect, the invention provides a method of modifying a targetpolynucleotide in a eukaryotic cell. In some embodiments, the methodcomprises allowing a CRISPR complex to bind to the target polynucleotideto effect cleavage of said target polynucleotide thereby modifying thetarget polynucleotide, wherein the CRISPR complex comprises a CRISPRenzyme complexed with a guide sequence hybridized to a target sequencewithin said target polynucleotide, wherein said guide sequence is linkedto a tracr mate sequence which in turn hybridizes to a tracr sequence.In some embodiments, said cleavage comprises cleaving one or two strandsat the location of the target sequence by said CRISPR enzyme. In someembodiments, said cleavage results in decreased transcription of atarget gene. In some embodiments, the method further comprises repairingsaid cleaved target polynucleotide by homologous recombination with anexogenous template polynucleotide, wherein said repair results in amutation comprising an insertion, deletion, or substitution of one ormore nucleotides of said target polynucleotide. In some embodiments,said mutation results in one or more amino acid changes in a proteinexpressed from a gene comprising the target sequence. In someembodiments, the method further comprises delivering one or more vectorsto said eukaryotic cell, wherein the one or more vectors driveexpression of one or more of: the CRISPR enzyme, the guide sequencelinked to the tracr mate sequence, and the tracr sequence. In someembodiments, said vectors are delivered to the eukaryotic cell in asubject. In some embodiments, said modifying takes place in saideukaryotic cell in a cell culture. In some embodiments, the methodfurther comprises isolating said eukaryotic cell from a subject prior tosaid modifying. In some embodiments, the method further comprisesreturning said eukaryotic cell and/or cells derived therefrom to saidsubject.

In one aspect, the invention provides a method of modifying expressionof a polynucleotide in a eukaryotic cell. In some embodiments, themethod comprises allowing a CRISPR complex to bind to the polynucleotidesuch that said binding results in increased or decreased expression ofsaid polynucleotide; wherein the CRISPR complex comprises a CRISPRenzyme complexed with a guide sequence hybridized to a target sequencewithin said polynucleotide, wherein said guide sequence is linked to atracr mate sequence which in turn hybridizes to a tracr sequence. Insome embodiments, the method further comprises delivering one or morevectors to said eukaryotic cells, wherein the one or more vectors driveexpression of one or more of: the CRISPR enzyme, the guide sequencelinked to the tracr mate sequence, and the tracr sequence.

In one aspect, the invention provides a method of generating a modeleukaryotic cell comprising a mutated disease gene. In some embodiments,a disease gene is any gene associated an increase in the risk of havingor developing a disease. In some embodiments, the method comprises (a)introducing one or more vectors into a eukaryotic cell, wherein the oneor more vectors drive expression of one or more of: a CRISPR enzyme, aguide sequence linked to a tracr mate sequence, and a tracr sequence;and (b) allowing a CRISPR complex to bind to a target polynucleotide toeffect cleavage of the target polynucleotide within said disease gene,wherein the CRISPR complex comprises the CRISPR enzyme complexed with(1) the guide sequence that is hybridizable to the target sequencewithin the target polynucleotide, and (2) the tracr mate sequence thatis hybridized to the tracr sequence, thereby generating a modeleukaryotic cell comprising a mutated disease gene. In some embodiments,said cleavage comprises cleaving one or two strands at the location ofthe target sequence by said CRISPR enzyme. In some embodiments, saidcleavage results in decreased transcription of a target gene. In someembodiments, the method further comprises repairing said cleaved targetpolynucleotide by homologous recombination with an exogenous templatepolynucleotide, wherein said repair results in a mutation comprising aninsertion, deletion, or substitution of one or more nucleotides of saidtarget polynucleotide. In some embodiments, said mutation results in oneor more amino acid changes in a protein expression from a genecomprising the target sequence.

In one aspect the invention provides for a method of selecting one ormore prokaryotic cell(s) by introducing one or more mutations in a genein the one or more prokaryotic cell (s), the method comprising:introducing one or more vectors into the prokaryotic cell (s), whereinthe one or more vectors drive expression of one or more of: a CRISPRenzyme, a guide sequence linked to a tracr mate sequence, a tracrsequence, and an editing template; wherein the editing templatecomprises the one or more mutations that abolish CRISPR enzyme cleavage;allowing homologous recombination of the editing template with thetarget polynucleotide in the cell(s) to be selected; allowing a CRISPRcomplex to bind to a target polynucleotide to effect cleavage of thetarget polynucleotide within said gene, wherein the CRISPR complexcomprises the CRISPR enzyme complexed with (1) the guide sequence thatis hybridizable to the target sequence within the target polynucleotide,and (2) the tracr mate sequence that is hybridized to the tracrsequence, wherein binding of the CRISPR complex to the targetpolynucleotide induces cell death, thereby allowing one or moreprokaryotic cell(s) in which one or more mutations have been introducedto be selected. In a preferred embodiment, the CRISPR enzyme is Cas9. Inanother aspect of the invention the cell to be selected may be aeukaryotic cell. Aspects of the invention allow for selection ofspecific cells without requiring a selection marker or a two-stepprocess that may include a counter-selection system.

In one aspect, the invention provides for methods of modifying a targetpolynucleotide in a eukaryotic cell. In some embodiments, the methodcomprises allowing a CRISPR complex to bind to the target polynucleotideto effect cleavage of said target polynucleotide thereby modifying thetarget polynucleotide, wherein the CRISPR complex comprises a CRISPRenzyme complexed with a guide sequence hybridized to a target sequencewithin said target polynucleotide, wherein said guide sequence is linkedto a tracr mate sequence which in turn hybridizes to a tracr sequence.

In other embodiments, this invention provides a method of modifyingexpression of a polynucleotide in a eukaryotic cell. The methodcomprises increasing or decreasing expression of a target polynucleotideby using a CRISPR complex that binds to the polynucleotide.

Where desired, to effect the modification of the expression in a cell,one or more vectors comprising a tracr sequence, a guide sequence linkedto the tracr mate sequence, a sequence encoding a CRISPR enzyme isdelivered to a cell. In some methods, the one or more vectors comprisesa regulatory element operably linked to an enzyme-coding sequenceencoding said CRISPR enzyme comprising a nuclear localization sequence;and a regulatory element operably linked to a tracr mate sequence andone or more insertion sites for inserting a guide sequence upstream ofthe tracr mate sequence. When expressed, the guide sequence directssequence-specific binding of a CRISPR complex to a target sequence in acell. Typically, the CRISPR complex comprises a CRISPR enzyme complexedwith (1) the guide sequence that is hybridizable to the target sequence,and (2) the tracr mate sequence that is hybridized to the tracrsequence.

In some methods, a target polynucleotide can be inactivated to effectthe modification of the expression in a cell. For example, upon thebinding of a CRISPR complex to a target sequence in a cell, the targetpolynucleotide is inactivated such that the sequence is not transcribed,the coded protein is not produced, or the sequence does not function asthe wild-type sequence does. For example, a protein or microRNA codingsequence may be inactivated such that the protein is not produced.

In certain embodiments, the CRISPR enzyme comprises one or moremutations D10A, E762A, H840A, N854A, N863A or D986A and/or the one ormore mutations is in a RuvCl or HNH domain of the CRISPR enzyme or is amutation as otherwise as discussed herein. In some embodiments, theCRISPR enzyme has one or more mutations in a catalytic domain, whereinwhen transcribed, the tracr mate sequence hybridizes to the tracrsequence and the guide sequence directs sequence-specific binding of aCRISPR complex to the target sequence, and wherein the enzyme furthercomprises a functional domain. In some embodiments, the mutated Cas9enzyme may be fused to a protein domain, e.g., such as a transcriptionalactivation domain. In one aspect, a transcriptional activation domain isVP64. In some embodiments, a transcription repression domains is KRAB.In some embodiments, a transcription repression domain is SID, orconcatemers of SID (i.e. SID4×). In some embodiments, an epigeneticmodifying enzyme is provided. In some embodiments, an activation domainis provided, which may be the P65 activation domain.

In some embodiments, the CRISPR enzyme is a type I or III CRISPR enzyme,preferably a type II CRISPR enzyme. This type II CRISPR enzyme may beany Cas enzyme. A Cas enzyme may be identified Cas9 as this can refer tothe general class of enzymes that share homology to the biggest nucleasewith multiple nuclease domains from the type II CRISPR system. Mostpreferably, the Cas9 enzyme is from, or is derived from, spCas9 orsaCas9. By derived, Applicants mean that the derived enzyme is largelybased, in the sense of having a high degree of sequence homology with, awildtype enzyme, but that it has been mutated (modified) in some way asdescribed herein.

The invention also provides a method of modifying a DNA duplex at alocus of interest in a cell, the method comprising delivering to thecell:

-   -   I. a first polynucleotide comprising:        -   (a) a first guide sequence capable of hybridizing to a first            target sequence,        -   (b) a first tracr mate sequence, and        -   (c) a first tracr sequence;    -   II. a second polynucleotide comprising:        -   (a) a second guide sequence capable of hybridizing to a            second target sequence,        -   (b) a second tracr mate sequence, and        -   (c) a second tracr sequence;    -   and    -   III. a third polynucleotide comprising a sequence encoding a        CRISPR enzyme and one or more nuclear localization sequences

wherein (a), (b) and (c) in said first and second polynucleotides arearranged in a 5′ to 3′ orientation;

wherein the first target sequence is on a first strand of the DNA duplexand the second target sequence is on the opposite strand of the DNAduplex, and when the first and second guide sequences are hybridized tosaid target sequences in the duplex, the 5′ ends of the firstpolynucleotide and the second polynucleotide are offset relative to eachother by at least one base pair of the duplex;

wherein when transcribed, the first and the second tracr mate sequenceshybridize to the first and second tracr sequences, respectively, and thefirst and the second guide sequences direct sequence-specific binding ofa first and a second CRISPR complex to the first and second targetsequences respectively,

wherein the first CRISPR complex comprises the CRISPR enzyme complexedwith (1) the first guide sequence that is hybridizable to the firsttarget sequence, and (2) the first tracr mate sequence that ishybridized to the first tracr sequence,

wherein the second CRISPR complex comprises the CRISPR enzyme complexedwith (1) the second guide sequence that is hybridizable to the secondtarget sequence, and (2) the second tracr mate sequence that ishybridized to the second tracr sequence,

and wherein said first strand of the DNA duplex is cleaved near saidfirst target sequence, and said opposite strand of the DNA duplex iscleaved near said second target sequence, resulting in a double strandbreak with a 5′ overhang or a 3′ overhang.

The invention also provides a method of modifying a DNA duplex at alocus of interest in a cell, the method comprising delivering to thecell a vector system comprising one or more vectors comprising:

-   -   I. a first polynucleotide sequence comprising a regulatory        element operably linked to        -   (a) a first guide sequence capable of hybridizing to a first            target sequence, and        -   (b) at least one or more tracr mate sequences,    -   II. a second polynucleotide sequence comprising a second        regulatory element operably linked to        -   (a) a second guide sequence capable of hybridizing to a            second target sequence, and        -   (b) at least one or more tracr mate sequences,    -   III. a third polynucleotide sequence comprising a third        regulatory element operably linked to a sequence encoding a        CRISPR enzyme, and    -   IV. a fourth polynucleotide sequence comprising a fourth        regulatory element operably linked to a tracr sequence,

wherein components I, II, III and IV are located on the same ordifferent vectors of the system

wherein the first target sequence is on a first strand of the DNA duplexand the second target sequence is on the opposite strand of the DNAduplex, and when the first and second guide sequences are hybridized tosaid target sequences in the duplex, the 5′ ends of the firstpolynucleotide and the second polynucleotide are offset relative to eachother by at least one base pair of the duplex;

wherein when transcribed, the first and the second tracr mate sequenceshybridize to a tracr sequence, and the first and the second guidesequences direct sequence-specific binding of a first and a secondCRISPR complex to the first and second target sequences respectively,

wherein the first CRISPR complex comprises the CRISPR enzyme complexedwith (1) the first guide sequence that is hybridizable to the firsttarget sequence, and (2) the first tracr mate sequence that ishybridized to a tracr sequence,

wherein the second CRISPR complex comprises the CRISPR enzyme complexedwith (1) the second guide sequence that is hybridizable to the secondtarget sequence, and (2) the second tracr mate sequence that ishybridized to a tracr sequence,

and wherein said first strand of the DNA duplex is cleaved near saidfirst target sequence, and said opposite strand of the DNA duplex iscleaved near said second target sequence, resulting in a double strandbreak with a 5′ overhang or a 3′ overhang.

In methods of the invention, the CRISPR enzyme advantageously is anickase enzyme, optionally a Cas9 enzyme comprising at least onemutation in a catalytic domain. For instance, there can be at least onemutation is in the RuvC domain and optionally is selected from the groupconsisting of D10A, E762A and D986A, or is in the HNH domain andoptionally is selected from the group consisting of H840A, N854A andN863A. In inventive methods, advantageously the offset between the 5′ends of the first polynucleotide and the second polynucleotide can begreater than −8 bp or −278 to +58 bp or −200 to +200 bp or up to or over100 bp or −4 to 20 bp or +23 bp or +16 or +20 or +16 to +20 bp or −3 to+18 bp; and it being understood that where appropriate one may use theterm nucleotide or nt for bp. Advantageously, in inventive methods thecleavage of said first strand and of said opposite strand of the DNAduplex occurs 3′ to a PAM (Protospacer adjacent motif) on each strand,and wherein said PAM on said first strand is separated from said PAM onsaid opposite strand by from 30 to 150 base pairs. In inventive methods,the overhang can be at most 200 bases, at most 100 bases, or at most 50bases; e.g., the overhang can be at least 1 base, at least 10 bases, atleast 15 bases, at least 26 bases or at least 30 bases; or, the overhangcan be between 34 and 50 bases or between 1 and 34 bases. Advantageouslyin inventive methods, any or all of the polynucleotide sequence encodingthe CRISPR enzyme, the first and the second guide sequence, the firstand the second tracr mate sequence or the first and the second tracrsequence, is/are RNA, and optionally wherein any or all of I, II and IIIare delivered via nanoparticles, exosomes, microvesicles, or a gene-gun.In inventive methods advantageously the first and second tracr matesequence can share 100% identity and/or the first and second tracrsequence share 100% identity. For instance, each of I, II and III can beprovided in a vector, optionally wherein each is provided in the same ora different vector. The locus of interest in inventive methods cancomprises a gene and wherein said method results in a change in theexpression of said gene, or in a change in the activity or function ofthe gene product. For instance, the gene product can be a protein,and/or wherein said change in expression, activity or function is areduction in said expression, activity or function. Inventive methodscan further comprise: (a) delivering to the cell a double-strandedoligodeoxynucleotide (dsODN) comprising overhangs complimentary to theoverhangs created by said double strand break, wherein said dsODN isintegrated into the locus of interest; or—(b) delivering to the cell asingle-stranded oligodeoxynucleotide (ssODN), wherein said ssODN acts asa template for homology directed repair of said double strand break.Inventive methods can be for the prevention or treatment of disease inan individual, optionally wherein said disease is caused by a defect insaid locus of interest. Inventive methods can be conducted in vivo inthe individual or ex vivo on a cell taken from the individual,optionally wherein said cell is returned to the individual.

The invention also provides a kit or composition comprising:

-   -   I. a first polynucleotide comprising:        -   (a) a first guide sequence capable of hybridizing to a first            target sequence,        -   (b) a first tracr mate sequence, and        -   (c) a first tracr sequence;    -   II. a second polynucleotide comprising:        -   (a) a second guide sequence capable of hybridizing to a            second target sequence,        -   (b) a second tracr mate sequence, and        -   (c) a second tracr sequence;    -   and    -   III. a third polynucleotide comprising a sequence encoding a        CRISPR enzyme and one or more nuclear localization sequences

wherein (a), (b) and (c) in said first and second polynucleotides arearranged in a 5′ to 3′ orientation;

wherein the first target sequence is on a first strand of a DNA duplexand the second target sequence is on the opposite strand of the DNAduplex, and when the first and second guide sequences are hybridized tosaid target sequences in the duplex, the 5′ ends of the firstpolynucleotide and the second polynucleotide are offset relative to eachother by at least one base pair of the duplex,

and optionally wherein each of I, II and III is provided in the same ora different vector.

The invention also provides use of a kit or composition according of theinvention in a method of the invention. The invention also provides useof a kit or composition of the invention in the manufacture of amedicament, optionally wherein said medicament is for the prevention ortreatment of a disease caused by a defect in said locus of interest.

It will be appreciated that the terms Cas and CRISPR enzyme aregenerally used herein interchangeably, unless otherwise apparent. Asmentioned above, many of the residue numberings used herein refer to theCas9 enzyme from the type II CRISPR locus in Streptococcus pyogenes.However, it will be appreciated that this invention includes many moreCas9s from other species of microbes, such as SpCas9, SaCa9, StlCas9 andso forth.

An example of a codon optimized sequence, in this instance optimized forhumans (i.e. being optimized for expression in humans), is providedherein, see the SaCas9 human codon optimized sequence. Whilst this ispreferred, it will be appreciated that other examples are possible andcodon optimization for a host species other than human, or for codonoptimization for specific organs such as the brain, is known.

In one aspect, delivery is in the form of a vector. In one aspect thevector may be a viral vector, such as a lenti- or baculo- or preferablyadeno-viral/adeno-associated viral vectors, but other means of deliveryare known (such as yeast systems, microvesicles, gene guns/means ofattaching vectors to gold nanoparticles) and are provided. A vector maymean not only a viral or yeast system (for instance, where the nucleicacids of interest may be operably linked to and under the control (interms of expression, such as to ultimately provide a processed RNA) apromoter), but also direct delivery of nucleic acids into a host cell.While in herein methods the vector may be a viral vector and this isadvantageously an AAV, other viral vectors as herein discussed can beemployed. For example, baculoviruses may be used for expression ininsect cells. These insect cells may, in turn be useful for producinglarge quantities of further vectors, such as AAV vectors adapted fordelivery of the present invention. Also envisaged is a method ofdelivering the present CRISPR enzyme comprising delivering to a cellmRNA encoding the CRISPR enzyme. It will be appreciated that the CRISPRenzyme is truncated, comprised of less than one thousand amino acids orless than four thousand amino acids, is a nuclease or nickase, iscodon-optimized comprises one or more mutations, and/or comprises achimeric CRISPR enzyme, or the other options as herein discussed. AAVviral vectors are preferred.

In certain embodiments, the target sequence is flanked or followed, atits 3′ end, by a PAM suitable for the CRISPR enzyme, typically a Cas andin particular a Cas9.

For example, a suitable PAM is 5′-NRG or 5′-NNGRR for SpCas9 or SaCas9enzymes (or derived enzymes), respectively.

It will be appreciated that SpCas9 or SaCas9 are those from or derivedfrom S. pyogenes or S. aureus Cas9.

Accordingly, it is an object of the invention to not encompass withinthe invention any previously known product, process of making theproduct, or method of using the product such that Applicants reserve theright and hereby disclose a disclaimer of any previously known product,process, or method. It is further noted that the invention does notintend to encompass within the scope of the invention any product,process, or making of the product or method of using the product, whichdoes not meet the written description and enablement requirements of theUSPTO (35 U.S.C. §112, first paragraph) or the EPO (Article 83 of theEPC), such that Applicants reserve the right and hereby disclose adisclaimer of any previously described product, process of making theproduct, or method of using the product.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. Patent law;e.g., they can mean “includes”, “included”, “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. Patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 shows a schematic model of the CRISPR system. The Cas9 nucleasefrom Streptococcus pyogenes (yellow) is targeted to genomic DNA by asynthetic guide RNA (sgRNA) consisting of a 20-nt guide sequence (blue)and a scaffold (red). The guide sequence base-pairs with the DNA target(blue), directly upstream of a requisite 5′-NGG protospacer adjacentmotif (PAM; magenta), and Cas9 mediates a double-stranded break (DSB) ˜3bp upstream of the PAM (red triangle).

FIG. 2A-F shows a schematic representation assay carried out to evaluatethe cleavage specificity of Cas9 form Streptococcus pyogenes. Singlebase pair mismatches between the guide RNA sequence and the target DNAare mapped against cleavage efficiency in %. FIG. 2C discloses SEQ IDNOS 255 and 256, respectively, in order of appearance. FIG. 2E disclosesSEQ ID NOS 257-259, respectively, in order of appearance. FIG. 2Fdiscloses SEQ ID NOS 260-264, respectively, in order of appearance.

FIG. 3A-D is a circular phylogenetic tree of Cas genes

FIG. 4A-F shows the linear phylogenetic analysis revealing five familiesof Cas9s, including three groups of large Cas9s (˜1400 amino acids) andtwo of small Cas9s (˜1100 amino acids).

FIG. 5 shows a graph representing the length distribution of Cas9orthologs.

FIG. 6A-M shows sequences where the mutation points are located withinthe SpCas9 gene. FIG. 6A-M discloses the nucleotide sequence as SEQ IDNO: 265 and the amino acid sequence as SEQ ID NO: 266.

FIG. 7A shows the Conditional Cas9, Rosa26 targeting vector map.

FIG. 7B shows the Constitutive Cas9, Rosa26 targeting vector map.

FIG. 8 shows a schematic of the important elements in the Constitutiveand Conditional Cas9 constructs.

FIG. 9 shows delivery and in vivo mouse brain Cas9 expression data.

FIG. 10 shows RNA delivery of Cas9 and chimeric RNA into cells (A)Delivery of a GFP reporter as either DNA or mRNA into Neuro-2A cells.(B) Delivery of Cas9 and chimeric RNA against the Icam2 gene as RNAresults in cutting for one of two spacers tested. (C) Delivery of Cas9and chimeric RNA against the F7 gene as RNA results in cutting for oneof two spacers tested.

FIG. 11 shows how DNA double-strand break (DSB) repair promotes geneediting. In the error-prone non-homologous end joining (NHEJ) pathway,the ends of a DSB are processed by endogenous DNA repair machineries andrejoined together, which can result in random insertion/deletion (indel)mutations at the site of junction. Indel mutations occurring within thecoding region of a gene can result in frame-shift and a premature stopcodon, leading to gene knockout. Alternatively, a repair template in theform of a plasmid or single-stranded oligodeoxynucleotides (ssODN) canbe supplied to leverage the homology-directed repair (HDR) pathway,which allows high fidelity and precise editing.

FIG. 12A-C shows anticipated results for HDR in HEK and HUES9 cells. (a)Either a targeting plasmid or an ssODN (sense or antisense) withhomology arms can be used to edit the sequence at a target genomic locuscleaved by Cas9 (red triangle). To assay the efficiency of HDR, weintroduced a HindIII site (red bar) into the target locus, which wasPCR-amplified with primers that anneal outside of the region ofhomology. Digestion of the PCR product with HindIII reveals theoccurrence of HDR events. (b) ssODNs, oriented in either the sense orthe antisense (s or a) direction relative to the locus of interest, canbe used in combination with Cas9 to achieve efficient HDR-mediatedediting at the target locus. A minimal homology region of 40 bp, andpreferably 90 bp, is recommended on either side of the modification (redbar). FIG. 12B discloses SEQ ID NOS 267-269, 267, 270, and 269,respectively, in order of appearance. (c) Example of the effect ofssODNs on HDR in the EMX1 locus is shown using both wild-type Cas9 andCas9 nickase (D10A). Each ssODN contains homology arms of 90 bp flankinga 12-bp insertion of two restriction sites.

FIG. 13A-C shows the repair strategy for Cystic Fibrosis delta F508mutation. FIG. 13A discloses the nucleotide sequence as SEQ ID NO: 271and the amino acid sequence as SEQ ID NO: 272. FIG. 13B discloses SEQ IDNO: 273. FIG. 13C discloses the nucleotide sequence as SEQ ID NO: 274and the amino acid sequence as SEQ ID NO: 275.

FIG. 14A-B shows a schematic of the GAA repeat expansion in FXN intron 1and (b) shows a schematic of the strategy adopted to excise the GAAexpansion region using the CRISPR/Cas system.

FIG. 15 shows a screen for efficient SpCas9 mediated targeting of Tet1-3and Dnmt1, 3a and 3b gene loci. Surveyor assay on DNA from transfectedN2A cells demonstrates efficient DNA cleavage by using different gRNAs.

FIG. 16 shows a strategy of multiplex genome targeting using a 2-vectorsystem in an AAV1/2 delivery system. Tet1-3 and Dnmt1, 3a and 3b gRNAunder the control of the U6 promoter. GFP-KASH under the control of thehuman synapsin promoter. Restriction sides shows simple gRNA replacementstrategy by subcloning. HA-tagged SpCas9 flanked by two nuclearlocalization signals (NLS) is shown. Both vectors are delivered into thebrain by AAV1/2 virus in a 1:1 ratio.

FIG. 17 shows verification of multiplex DNMT targeting vector #1functionality using Surveyor assay. N2A cells were co-transfected withthe DNMT targeting vector #1 (+) and the SpCas9 encoding vector fortesting SpCas9 mediated cleavage of DNMTs genes family loci. gRNA only(−) is negative control. Cells were harvested for DNA purification anddownstream processing 48 h after transfection.

FIG. 18 shows verification of multiplex DNMT targeting vector #2functionality using Surveyor assay. N2A cells were co-transfected withthe DNMT targeting vector #1 (+) and the SpCas9 encoding vector fortesting SpCas9 mediated cleavage of DNMTs genes family loci. gRNA only(−) is negative control. Cells were harvested for DNA purification anddownstream processing 48 h after transfection.

FIG. 19 shows schematic overview of short promoters and short polyAversions used for HA-SpCas9 expression in vivo. Sizes of the encodingregion from L-ITR to R-ITR are shown on the right.

FIG. 20 shows schematic overview of short promoters and short polyAversions used for HA-SaCas9 expression in vivo. Sizes of the encodingregion from L-ITR to R-ITR are shown on the right.

FIG. 21 shows expression of SpCas9 and SaCas9 in N2A cells.Representative Western blot of HA-tagged SpCas9 and SaCas9 versionsunder the control of different short promoters and with or short polyA(spA) sequences. Tubulin is loading control. mCherry (mCh) is atransfection control. Cells were harvested and further processed forWestern blotting 48 h after transfection.

FIG. 22 shows screen for efficient SaCas9 mediated targeting of Tet3gene locus. Surveyor assay on DNA from transfected N2A cellsdemonstrates efficient DNA cleavage by using different gRNAs with NNGGGTPUM sequence. GFP transfected cells and cells expressing only SaCas9 arecontrols.

FIG. 23 shows expression of HA-SaCas9 in the mouse brain. Animals wereinjected into dentate gyri with virus driving expression of HA-SaCas9under the control of human Synapsin promoter. Animals were sacrificed 2weeks after surgery. HA tag was detected using rabbit monoclonalantibody C29F4 (Cell Signaling). Cell nuclei stained in blue with DAPIstain.

FIG. 24 shows expression of SpCas9 and SaCas9 in cortical primaryneurons in culture 7 days after transduction. Representative Westernblot of HA-tagged SpCas9 and SaCas9 versions under the control ofdifferent promoters and with bgh or short polyA (spA) sequences. Tubulinis loading control.

FIG. 25 shows LIVE/DEAD stain of primary cortical neurons 7 days aftertransduction with AAV1 particles carrying SpCas9 with differentpromoters and multiplex gRNAs constructs (example shown on the lastpanel for DNMTs). Neurons after AAV transduction were compared withcontrol untransduced neurons. Red nuclei indicate permeabilized, deadcells (second line of panels). Live cells are marked in green color(third line of panels).

FIG. 26 shows LIVE/DEAD stain of primary cortical neurons 7 days aftertransduction with AAV1 particles carrying SaCas9 with differentpromoters. Red nuclei indicate permeabilized, dead cells (second line ofpanels). Live cells are marked in green color (third line of panels).

FIG. 27 shows comparison of morphology of neurons after transductionwith AAV1 virus carrying SpCas9 and gRNA multiplexes for TETs and DNMTsgenes loci. Neurons without transduction are shown as a control.

FIG. 28 shows verification of multiplex DNMT targeting vector #1functionality using Surveyor assay in primary cortical neurons. Cellswere co-transduced with the DNMT targeting vector #1 and the SpCas9viruses with different promoters for testing SpCas9 mediated cleavage ofDNMTs genes family loci.

FIG. 29 shows in vivo efficiency of SpCas9 cleavage in the brain. Micewere injected with AAV1/2 virus carrying gRNA multiplex targeting DNMTfamily genes loci together with SpCas9 viruses under control of 2different promoters: mouse Mecp2 and rat Map1b. Two weeks afterinjection brain tissue was extracted and nuclei were prepped and sortedusing FACS, based on the GFP expression driven by Synapsin promoter fromgRNA multiplex construct. After gDNA extraction Surveyor assay wasrun. + indicates GFP positive nuclei and − control, GFP-negative nucleifrom the same animal. Numbers on the gel indicate assessed SpCas9efficiency.

FIG. 30 shows purification of GFP-KASH labeled cell nuclei fromhippocampal neurons. The outer nuclear membrane (ONM) of the cellnuclear membrane is tagged with a fusion of GFP and the KASH proteintransmembrane domain. Strong GFP expression in the brain after one weekof stereotactic surgery and AAV1/2 injection. Density gradientcentrifugation step to purify cell nuclei from intact brain. Purifiednuclei are shown. Chromatin stain by Vybrant® DyeCycle™ Ruby Stain isshown in red, GFP labeled nuclei are green. Representative FACS profileof GFP+ and GFP− cell nuclei (Magenta: Vybrant® DyeCycle™ Ruby Stain,Green: GFP).

FIG. 31 shows efficiency of SpCas9 cleavage in the mouse brain. Micewere injected with AAV1/2 virus carrying gRNA multiplex targeting TETfamily genes loci together with SpCas9 viruses under control of 2different promoters: mouse Mecp2 and rat Map1b. Three weeks afterinjection brain tissue was extracted, nuclei were prepped and sortedusing FACS, based on the GFP expression driven by Synapsin promoter fromgRNA multiplex construct. After gDNA extraction Surveyor assay wasrun. + indicates GFP positive nuclei and − control, GFP-negative nucleifrom the same animal. Numbers on the gel indicate assessed SpCas9efficiency.

FIG. 32 shows GFP-KASH expression in cortical neurons in culture.Neurons were transduced with AAV1 virus carrying gRNA multiplexconstructs targeting TET genes loci. The strongest signal localizearound cells nuclei due to KASH domain localization.

FIG. 33 shows (top) a list of spacing (as indicated by the pattern ofarrangement for two PAM sequences) between pairs of guide RNAs. Onlyguide RNA pairs satisfying patterns 1, 2, 3, 4 exhibited indels whenused with SpCas9(D10A) nickase. (bottom) Gel images showing thatcombination of SpCas9(D10A) with pairs of guide RNA satisfying patterns1, 2, 3, 4 led to the formation of indels in the target site. FIG. 33discloses SEQ ID NOS 276-292, respectively, in order of appearance.

FIG. 34 shows a list of U6 reverse primer sequences used to generateU6-guide RNA expression casssettes. Each primer needs to be paired withthe U6 forward primer “gcactgagggcctatttcccatgattc” (SEQ ID NO: 1) togenerate amplicons containing U6 and the desired guide RNA. FIG. 34discloses SEQ ID NOS 293-339 and 295, respectively, in order ofappearance.

FIG. 35 shows a Genomic sequence map from the human Emx1 locus showingthe locations of the 24 patterns listed in FIG. 33. FIG. 35 disclosesthe nucleotide sequence as SEQ ID NO: 340 and the amino acid sequencesas 341-344, respectively, in order of appearance.

FIG. 36 shows on (right) a gel image indicating the formation of indelsat the target site when variable 5′ overhangs are present after cleavageby the Cas9 nickase targeted by different pairs of guide RNAs. on (left)a table indicating the lane numbers of the gel on the right and variousparameters including identifying the guide RNA pairs used and the lengthof the 5′ overhang present following cleavage by the Cas9 nickase.

FIG. 37 shows a Genomic sequence map from the human Emx1 locus showingthe locations of the different pairs of guide RNAs that result in thegel patterns of FIG. 36 (right) and which are further described inExample 30. FIG. 37 discloses the nucleotide sequence as SEQ ID NO: 340and the amino acid sequences as 341-344, respectively, in order ofappearance.

FIG. 38A-B shows the effect of guide sequence extension on Cas9 activity(A) Schematic showing Cas9 with matching or mismatching sgRNA sequencestargeting a locus (target 1) within the human EMX1 gene. FIG. 38Adiscloses SEQ ID NOS 345-348, respectively, in order of appearance. (B)SURVEYOR assay gel showing comparable modification of target 1 by sgRNAsbearing 20- and 30-nt long guide sequences. (C) Northern blot showingthat extended sgRNAs are largely reverted to 20-nt guide-length sgRNAsin HEK 293FT cells.

FIG. 39A-C shows that double nicking facilitates efficient genomeediting in human cells (A) Schematic illustrating DNA double-strandedbreaks using a pair of sgRNAs guiding Cas9 D10A nickases (Cas9n). TheD10A mutation renders Cas9 able to cleave only the strand complementaryto the sgRNA; a pair of sgRNA-Cas9n complexes can nick both strandssimultaneously. sgRNA offset is defined as the distance between thePAM-distal (5′) ends of the guide sequence of a given sgRNA pair;positive offset requires the sgRNA complementary to the top strand(sgRNA a) to be 5′ of the sgRNA complementary to the bottom strand(sgRNA b), which always creates a 5′-overhang. (B) Efficiency of doublenicking induced NHEJ as a function of the offset distance between twosgRNAs. Sequences for all sgRNAs used can be found in Table 51. (n=3;error bars show mean±s.e.m.) (C) Representative sequences of the humanEMX1 locus targeted by Cas9n. sgRNA target sites and PAMs are indicatedby blue and magenta bars respectively. Below, selected sequences showingrepresentative indels. See also FIGS. 44 and 45. FIG. 39C discloses SEQID NOS 349-357, respectively, in order of appearance.

FIG. 40A-F shows that double nicking facilitates efficient genomeediting in human cells (A) Schematic illustrating Cas9n double nicking(red arrows) the human EMX1 locus. Five off-target loci with sequencehomology to EMX1 target 1 were selected to screen for Cas9n specificity.FIG. 40A discloses SEQ ID NOS 358-363, respectively, in order ofappearance. (B) On-target modification rate by Cas9n and a pair ofsgRNAs is comparable to those mediated by wildtype Cas9 and singlesgRNAs (left panel). Cas9-sgRNA 1 complexes generate significantoff-target mutagenesis, while no off-target locus modification isdetected with Cas9n (right panel). (C) Five off-target loci of sgRNA 1are examined for indel modifications by deep sequencing of transfectedHEK 293FT cells. (n=3, error bars show mean±s.e.m.) (D) Specificitycomparison of Cas9n with double nicking and wildtype Cas9 with sgRNA 1alone at the off-target sites. Specificity ratio is calculated ason-target/off-target modification rates. (n=3; error bars showmean±s.e.m.) (E, F) Double nicking minimizes off-target modification attwo additional human VEGFA loci while maintaining high specificity(on/off target modification ratio; n=3, error bars show mean±s.e.m.).

FIG. 41A-D shows that double nicking allows insertion into the genomevia HDR in human cells (A) Schematic illustrating HDR mediated via asingle stranded oligodeoxynucleotide (ssODN) template at a DSB createdby a pair of Cas9n enzymes. A 12-nt sequence (red), including a HindIIIrestriction site, is inserted into the EMX1 locus at the position markedby the gray dashed lines; distances of Cas9n-mediated nicks from the HDRinsertion site is indicated on top in italics. FIG. 41A discloses SEQ IDNOS 364-365, respectively, in order of appearance. (B) Restrictiondigest assay gel showing successful insertion of HindIII cleavage sitesby double nicking-mediated HDR in HEK 293FT cells. Upper bands areunmodified template; lower bands are HindIII cleavage product. (C)Double nicking promotes HDR in the HUES62 human embryonic stem cellline. HDR frequencies are determined by deep sequencing. (n=3; errorbars show mean±s.e.m.). (D) HDR efficiency depends on the configurationof Cas9 or Cas9n-mediated nicks. HDR is facilitated when a nick occursnear the center of the ssODN homology arm (HDR insertion site) leadingto a 5′-resulting overhang. Nicking configurations are annotated withposition and strand (red arrows) and length of overhang (black lines)(left panel). The distance (bp) of each nick from the HDR insertion siteis indicated at the end of the black lines in italics, and the positionsof the sgRNAs are illustrated in bold on the schematic of the EMX1locus. HDR efficiency mediated by double nicking with paired sgRNAs (toppanel) or single sgRNAs with either Cas9 or Cas9n are shown (bottompanel, FIG. 45; n=3, error bars show mean±s.e.m.).

FIG. 42A-B shows that multiplexed nicking facilitates non-HR mediatedgene integration and genomic deletions (A) Schematic showing insertionof a double-stranded oligodeoxynucleotide (dsODN) donor fragment bearingoverhangs complementary to 5′ overhangs created by Cas9 double nicking.The dsODN was designed to remove the native EMX1 stop codon and containsa HA tag, 3× FLAG tag, HindIII restriction site, Myc epitope tag, and astop codon in frame, totaling 148 bp. Successful insertion was verifiedby Sanger sequencing as shown (1/37 clones screened). Amino acidtranslation of the modified locus is shown below the DNA sequence. FIG.42A discloses SEQ ID NOS 366-368, respectively, in order of appearance.(B) Co-delivery of four sgRNAs with Cas9n generate long-range genomicdeletions in the DYRK1A locus (from 0.5 kb up to 6 kb). Deletion wasdetected using primers (Table S6) spanning the target region.

FIG. 43A-B shows that Cas9 double nicking mediates efficient indelformation in mouse embryos (A) Schematic illustrating Cas9n doublenicking the mouse Mecp2 locus. Representative indels are shown for mouseblastocysts co-injected with in vitro transcribed Cas9n-encoding mRNAand sgRNA pairs matching targets 92 and 93. FIG. 43A discloses SEQ IDNOS 369-379, respectively, in order of appearance. (B) Efficientblastocyst modification is achieved at multiple concentrations of sgRNAs(1.5 to 50 ng/uL) and wildtype Cas9 or Cas9n (ng/uL to 100 ng/uL).

FIG. 44A-F shows a list of sgRNA pairs used with Cas9 nickase (D10A) toidentify the optimal target site spacing for double nicking acrossmultiple genes, related to FIG. 39. N.D.: not detected. N.T.: nottested. Left sgRNA target site guide sequences. EMX1 disclosed as SEQ IDNOS 380, 381, 381, 382, 383, 384, 385, 385, 386, 380, 380, 387, 387,386, 386, 385, 387, 386, 387, 386, 388, 389, 386, and 387. DYRK1Adisclosed as SEQ ID NOS 390, 391, 392, 391, 393, 394, 395, 396, 397,390, 398, 399, 400, 401, 402, 403, 392, 394, 394, 404, 403 and 405.GRIN2B disclosed as SEQ ID NOS 406, 407, 408, 409, 410, 411, 412, 413,414, 415, 412, 416, 417, 418, 419, 413, 420, 412, 413, 421, 414, 422,421 and 422. MeCP2 disclosed as SEQ ID NOS 473 and 474. VEGFA disclosedas SEQ ID NOS 475 and 476. Right sgRNA target site guide sequences. EMX1disclosed as SEQ ID NOS 383, 423, 424, 425, 387, 385, 426, 382, 384,427, 424, 384, 428, 382, 429, 430, 382, 431, 430, 427, 432, 432, 426 and426. DYRK1A disclosed as SEQ ID NOS 433, 433, 434, 435, 436, 433, 437,435, 438, 439, 440, 399, 436, 436, 437, 441, 436, 440, 435, 440, 436 and440. GRIN2B disclosed as SEQ ID NOS 442, 443, 444, 445, 446, 447, 448,448, 446, 445, 449, 449, 450, 451, 452, 449, 453, 447, 447, 454, 455,456, 456 and 457. MeCP2 disclosed as SEQ ID NOS 477 and 478. VEGFAdisclosed as SEQ ID NOS 479 and 480. All sequences identified in orderof appearance.

FIG. 45A-D shows a list of sgRNAs used in Example 31. Related to FIG.39. Guide sequences. EMX1 disclosed as SEQ ID NOS 423, 458, 459, 383,387, 388, 384, 428, 385, 382, 429, 431, 425, 430, 380, 427, 424, 432,426, 460, 461, 462, 381, 463, 464, 465 and 461. DYRK1A disclosed as SEQID NOS 466, 399, 400, 436, 402, 437, 390, 391, 392, 393, 394, 395, 396,397, 398, 401, 403, 404, 405, 433, 434, 435, 438, 439, 440, 441, 418,451, 419, 452, 417, 450, 420 and 453. GRIN2B disclosed as SEQ ID NOS467, 468, 469, 449, 470, 456, 471, 472, 406, 407, 408, 409, 410, 411,412, 413, 414, 416, 421, 422, 442, 443, 444, 445, 446, 447, 448, 454,455 and 457. MeCP2 disclosed as SEQ ID NOS 473, 477, 474 and 478. VEGFAdisclosed as SEQ ID NOS 475, 479, 476 and 480. All sequences identifiedin order of appearance.

DETAILED DESCRIPTION OF THE INVENTION

With respect to general information on CRISPR-Cas Systems: Reference ismade to U.S. provisional applications 61/758,468; 61/802,174;61/806,375; 61/814,263; 61/819,803 and 61/828,130, filed on Jan. 30,2013; Mar. 15, 2013; Mar. 28, 2013; Apr. 20, 2013; May 6, 2013 and May28, 2013 respectively. Reference is also made to U.S. provisional patentapplications 61/836,123, 61/847,537, 61/862,355 and 61/871,301, filed onJun. 17, 2013; Jul. 17, 2013, Aug. 5, 2013 and Aug. 28, 2013respectively. Reference is further made to U.S. provisional patentapplications 61/736,527 and 61/748,427 filed on Dec. 12, 2012 and Jan.2, 2013, respectively. Reference is additionally made to U.S.provisional patent application 61/791,409, filed on Mar. 15, 2013.Reference is also made to U.S. provisional patent application61/799,800, filed Mar. 15, 2013. Reference is also made to U.S.provisional patent applications 61/835,931, 61/835,936, 61/836,127,61/836,101, 61/836,123, 61/836,080, and 61/835,973 each filed Jun. 17,2013. Each of these applications, and all documents cited therein orduring their prosecution (“appin cited documents”) and all documentscited or referenced in the appin cited documents, together with anyinstructions, descriptions, product specifications, and product sheetsfor any products mentioned therein or in any document therein andincorporated by reference herein, are hereby incorporated herein byreference, and may be employed in the practice of the invention. Alldocuments (e.g., these applications and the appin cited documents) areincorporated herein by reference to the same extent as if eachindividual document was specifically and individually indicated to beincorporated by reference.

Also with respect to general information on CRISPR-Cas Systems, mentionis made of:

-   -   Multiplex genome engineering using CRISPR/Cas systems. Cong, L.,        Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P.        D., Wu, X., Jiang, W., Marraffini, L. A., & Zhang, F. Science        February 15; 339(6121):819-23 (2013);    -   RNA guided editing of bacterial genomes using CRISPR-Cas        systems. Jiang W., Bikard D., Cox D., Zhang F, Marraffini L A.        Nat Biotechnol March; 31(3):233-9 (2013);    -   One-Step Generation of Mice Carrying Mutations in Multiple Genes        by CRISPR/Cas-Mediated Genome Engineering. Wang H., Yang H.,        Shivalila C S., Dawlaty M M., Cheng A W., Zhang F., Jaenisch R.        Cell May 9; 153(4):910-8 (2013);    -   Optical control of mammalian endogenous transcription and        epigenetic states. Konermann S, Brigham M D, Trevino A E, Hsu P        D, Heidenreich M, Cong L, Platt R J, Scott D A, Church G M,        Zhang F. Nature. 2013 Aug. 22; 500(7463):472-6. doi:        10.1038/Nature12466. Epub 2013 Aug. 23;    -   Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome        Editing Specificity. Ran, F A., Hsu, P D., Lin, C Y.,        Gootenberg, J S., Konermann, S., Trevino, A E., Scott, D A.,        Inoue, A., Matoba, S., Zhang, Y., & Zhang, F. Cell August 28.        pii: S0092-8674(13)01015-5. (2013);    -   DNA targeting specificity of RNA guided Cas9 nucleases. Hsu, P.,        Scott, D., Weinstein, J., Ran, F A., Konermann, S., Agarwala,        V., Li, Y., Fine, E., Wu, X., Shalem, O., Cradick, T J.,        Marraffini, L A., Bao, G., & Zhang, F. Nat Biotechnol        doi:10.1038/nbt.2647 (2013);    -   Genome engineering using the CRISPR-Cas9 system. Ran, F A., Hsu,        P D., Wright, J., Agarwala, V., Scott, D A., Zhang, F. Nature        Protocols November; 8(11):2281-308. (2013);    -   Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells.        Shalem, O., Sanjana, N E., Hartenian, E., Shi, X., Scott, D A.,        Mikkelson, T., Heckl, D., Ebert, B L., Root, D E., Doench, J G.,        Zhang, F. Science December 12. (2013). [Epub ahead of print];    -   Crystal structure of cas9 in complex with guide RNA and target        DNA. Nishimasu, H., Ran, F A., Hsu, P D., Konermann, S.,        Shehata, S I., Dohmae, N., Ishitani, R., Zhang, F., Nureki, O.        Cell February 27. (2014). 156(5):935-49;    -   Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian        cells. Wu X., Scott D A., Kriz A J., Chiu A C., Hsu P D., Dadon        D B., Cheng A W., Trevino A E., Konermann S., Chen S., Jaenisch        R., Zhang F., Sharp P A. Nat Biotechnol. (2014) Apr. 20. doi:        10.1038/nbt.2889, and    -   Development and Applications of CRISPR-Cas9 for Genome        Engineering, Hsu et al, Cell 157, 1262-1278 (Jun. 5, 2014) (Hsu        2014),        each of which is incorporated herein by reference, and discussed        briefly below:    -   Cong et al. engineered type II CRISPR/Cas systems for use in        eukaryotic cells based on both Streptococcus thermophilus Cas9        and also Streptoccocus pyogenes Cas9 and demonstrated that Cas9        nucleases can be directed by short RNAs to induce precise        cleavage of DNA in human and mouse cells. Their study further        showed that Cas9 as converted into a nicking enzyme can be used        to facilitate homology-directed repair in eukaryotic cells with        minimal mutagenic activity. Additionally, their study        demonstrated that multiple guide sequences can be encoded into a        single CRISPR array to enable simultaneous editing of several at        endogenous genomic loci sites within the mammalian genome,        demonstrating easy programmability and wide applicability of the        RNA-guided nuclease technology. This ability to use RNA to        program sequence specific DNA cleavage in cells defined a new        class of genome engineering tools. These studies further showed        that other CRISPR loci are likely to be transplantable into        mammalian cells and can also mediate mammalian genome cleavage.        Importantly, it can be envisaged that several aspects of the        CRISPR/Cas system can be further improved to increase its        efficiency and versatility.    -   Jiang et al. used the clustered, regularly interspaced, short        palindromic repeats (CRISPR)-associated Cas9 endonuclease        complexed with dual-RNAs to introduce precise mutations in the        genomes of Streptococcus pneumoniae and Escherichia coli. The        approach relied on dual-RNA:Cas9-directed cleavage at the        targeted genomic site to kill unmutated cells and circumvents        the need for selectable markers or counter-selection systems.        The study reported reprogramming dual-RNA:Cas9 specificity by        changing the sequence of short CRISPR RNA (crRNA) to make        single- and multinucleotide changes carried on editing        templates. The study showed that simultaneous use of two crRNAs        enabled multiplex mutagenesis. Furthermore, when the approach        was used in combination with recombineering, in S. pneumoniae,        nearly 100% of cells that were recovered using the described        approach contained the desired mutation, and in E. coli, 65%        that were recovered contained the mutation.    -   Konermann et al. addressed the need in the art for versatile and        robust technologies that enable optical and chemical modulation        of DNA-binding domains based CRISPR Cas9 enzyme and also        Transcriptional Activator Like Effectors    -   As discussed in the present specification, the Cas9 nuclease        from the microbial CRISPR-Cas system is targeted to specific        genomic loci by a 20 nt guide sequence, which can tolerate        certain mismatches to the DNA target and thereby promote        undesired off-target mutagenesis. To address this, Ran et al.        described an approach that combined a Cas9 nickase mutant with        paired guide RNAs to introduce targeted double-strand breaks.        Because individual nicks in the genome are repaired with high        fidelity, simultaneous nicking via appropriately offset guide        RNAs is required for double-stranded breaks and extends the        number of specifically recognized bases for target cleavage. The        authors demonstrated that using paired nicking can reduce        off-target activity by 50- to 1,500-fold in cell lines and to        facilitate gene knockout in mouse zygotes without sacrificing        on-target cleavage efficiency. This versatile strategy enables a        wide variety of genome editing applications that require high        specificity.    -   Hsu et al. characterized SpCas9 targeting specificity in human        cells to inform the selection of target sites and avoid        off-target effects. The study evaluated >700 guide RNA variants        and SpCas9-induced indel mutation levels at >100 predicted        genomic off-target loci in 293T and 293FT cells. The authors        that SpCas9 tolerates mismatches between guide RNA and target        DNA at different positions in a sequence-dependent manner,        sensitive to the number, position and distribution of        mismatches. The authors further showed that SpCas9-mediated        cleavage is unaffected by DNA methylation and that the dosage of        SpCas9 and sgRNA can be titrated to minimize off-target        modification. Additionally, to facilitate mammalian genome        engineering applications, the authors reported providing a        web-based software tool to guide the selection and validation of        target sequences as well as off-target analyses.    -   Ran et al. described a set of tools for Cas9-mediated genome        editing via non-homologous end joining (NHEJ) or        homology-directed repair (HDR) in mammalian cells, as well as        generation of modified cell lines for downstream functional        studies. To minimize off-target cleavage, the authors further        described a double-nicking strategy using the Cas9 nickase        mutant with paired guide RNAs. The protocol provided by the        authors experimentally derived guidelines for the selection of        target sites, evaluation of cleavage efficiency and analysis of        off-target activity. The studies showed that beginning with        target design, gene modifications can be achieved within as        little as 1-2 weeks, and modified clonal cell lines can be        derived within 2-3 weeks.    -   Shalem et al. described a new way to interrogate gene function        on a genome-wide scale. Their studies showed that delivery of a        genome-scale CRISPR-Cas9 knockout (GeCKO) library targeted        18,080 genes with 64,751 unique guide sequences enabled both        negative and positive selection screening in human cells. First,        the authors showed use of the GeCKO library to identify genes        essential for cell viability in cancer and pluripotent stem        cells. Next, in a melanoma model, the authors screened for genes        whose loss is involved in resistance to vemurafenib, a        therapeutic that inhibits mutant protein kinase BRAF. Their        studies showed that the highest-ranking candidates included        previously validated genes NF1 and MED12 as well as novel hits        NF2, CUL3, TADA2B, and TADA1. The authors observed a high level        of consistency between independent guide RNAs targeting the same        gene and a high rate of hit confirmation, and thus demonstrated        the promise of genome-scale screening with Cas9.    -   Nishimasu et al. reported the crystal structure of Streptococcus        pyogenes Cas9 in complex with sgRNA and its target DNA at 2.5 A°        resolution. The structure revealed a bilobed architecture        composed of target recognition and nuclease lobes, accommodating        the sgRNA:DNA heteroduplex in a positively charged groove at        their interface. Whereas the recognition lobe is essential for        binding sgRNA and DNA, the nuclease lobe contains the HNH and        RuvC nuclease domains, which are properly positioned for        cleavage of the complementary and non-complementary strands of        the target DNA, respectively. The nuclease lobe also contains a        carboxyl-terminal domain responsible for the interaction with        the protospacer adjacent motif (PAM). This high-resolution        structure and accompanying functional analyses have revealed the        molecular mechanism of RNA-guided DNA targeting by Cas9, thus        paving the way for the rational design of new, versatile        genome-editing technologies.    -   Wu et al. mapped genome-wide binding sites of a catalytically        inactive Cas9 (dCas9) from Streptococcus pyogenes loaded with        single guide RNAs (sgRNAs) in mouse embryonic stem cells        (mESCs). The authors showed that each of the four sgRNAs tested        targets dCas9 to between tens and thousands of genomic sites,        frequently characterized by a 5-nucleotide seed region in the        sgRNA and an NGG protospacer adjacent motif (PAM). Chromatin        inaccessibility decreases dCas9 binding to other sites with        matching seed sequences; thus 70% of off-target sites are        associated with genes. The authors showed that targeted        sequencing of 295 dCas9 binding sites in mESCs transfected with        catalytically active Cas9 identified only one site mutated above        background levels. The authors proposed a two-state model for        Cas9 binding and cleavage, in which a seed match triggers        binding but extensive pairing with target DNA is required for        cleavage.    -   Hsu 2014 is a review article that discusses generally        CRISPR-Cas9 history from yogurt to genome editing, including        genetic screening of cells, that is in the information, data and        findings of the applications in the lineage of this        specification filed prior to Jun. 5, 2014. The general teachings        of Hsu 2014 do not involve the specific models, animals of the        instant specification.

The invention relates to the engineering and optimization of systems,methods and compositions used for the control of gene expressioninvolving sequence targeting, such as genome perturbation orgene-editing, that relate to the CRISPR-Cas system and componentsthereof. In advantageous embodiments, the Cas enzyme is Cas9.

An advantage of the present methods is that the CRISPR system avoidsoff-target binding and its resulting side effects. This is achievedusing systems arranged to have a high degree of sequence specificity forthe target DNA.

Cas9 optimization may be used to enhance function or to develop newfunctions, one can generate chimeric Cas9 proteins. Examples that theApplicants have generated are provided in Example 6. Chimeric Cas9proteins can be made by combining fragments from different Cas9homologs. For example, two example chimeric Cas9 proteins from the Cas9sdescribed herein. For example, Applicants fused the N-term of StlCas9(fragment from this protein is in bold) with C-term of SpCas9. Thebenefit of making chimeric Cas9s include any or all of:

reduced toxicity;improved expression in eukaryotic cells;enhanced specificity;reduced molecular weight of protein, make protein smaller by combiningthe smallest domains from different Cas9 homologs; and/oraltering the PAM sequence requirement.

The Cas9 may be used as a generic DNA binding protein. For example, andas shown in Example 7, Applicants used Cas9 as a generic DNA bindingprotein by mutating the two catalytic domains (D10 and H840) responsiblefor cleaving both strands of the DNA target. In order to upregulate genetranscription at a target locus Applicants fused the transcriptionalactivation domain (VP64) to Cas9. Other transcriptional activationdomains are known. As shown in Example 11, transcriptional activation ispossible. As also shown in Example 11, gene repression (in this case ofthe beta-catenin gene) is possible using a Cas9 repressor (DNA-bindingdomain) that binds to the target gene sequence, thus repressing itsactivity.

Transgenic animals are also provided. Preferred examples include animalscomprising Cas9, in terms of polynucleotides encoding Cas9 or theprotein itself. Mice, rats and rabbits are preferred. To generatetransgenic mice with the constructs, as exemplified herein one mayinject pure, linear DNA into the pronucleus of a zygote from a pseudopregnant female, e.g. a CB56 female. Founders may then be identified areidentified, genotyped, and backcrossed to CB57 mice. The constructs maythen be cloned and optionally verified, for instance by Sangersequencing. Knock outs are envisaged where for instance one or moregenes are knocked out in a model. However, are knockins are alsoenvisaged (alone or in combination). An example Knock in Cas9 mouse wasgenerated and this is exemplified, but Cas9 knockins are preferred. Togenerate a Cas9 knock in mice one may target the same constitutive andconditional constructs to the Rosa26 locus, as described herein (FIGS.7A-B and 8). That the CRISPR-Cas system is able to be employed ingenerating transgenic mice is also provided in the manuscript “One-stepgeneration of mice carrying mutations in multiple genes byCRISPR/Cas-mediated genome engineering.” by Wang et al. Cell. 2013 May9; 153(4):910-8. doi: 10.1016/j.cell.2013.04.025. Epub 2013 May 2.,incorporated by reference in its entirety, wherein it is demonstratedthat CRISPR/Cas mediated gene editing allows for the simultaneousdisruption of five genes in mouse embryonic stem cells with highefficiency.

Utility of the conditional Cas9 mouse: Applicants have shown in 293cells that the Cas9 conditional expression construct can be activated byco-expression with Cre. Applicants also show that the correctly targetedR1 mESCs can have active Cas9 when Cre is expressed. Because Cas9 isfollowed by the P2A peptide cleavage sequence and then EGFP Applicantsidentify successful expression by observing EGFP. Applicants have shownCas9 activation in mESCs. This same concept is what makes theconditional Cas9 mouse so useful. Applicants may cross their conditionalCas9 mouse with a mouse that ubiquitously expresses Cre (ACTB-Cre line)and may arrive at a mouse that expresses Cas9 in every cell. It shouldonly take the delivery of chimeric RNA to induce genome editing inembryonic or adult mice. Interestingly, if the conditional Cas9 mouse iscrossed with a mouse expressing Cre under a tissue specific promoter,there should only be Cas9 in the tissues that also express Cre. Thisapproach may be used to edit the genome in only precise tissues bydelivering chimeric RNA to the same tissue.

As mentioned above, transgenic animals are also provided, as aretransgenic plants, especially crops and algae. The transgenic may beuseful in applications outside of providing a disease model. These mayinclude food of feed production through expression of, for instance,higher protein, carbohydrate, nutrient or vitamins levels than wouldnormally be seen in the wildtype. In this regard, transgenic plants,especially pulses and tubers, and animals, especially mammals such aslivestock (cows, sheep, goats and pigs), but also poultry and edibleinsects, are preferred.

Transgenic algae or other plants such as rape may be particularly usefulin the production of vegetable oils or biofuels such as alcohols(especially methanol and ethanol), for instance. These may be engineeredto express or overexpress high levels of oil or alcohols for use in theoil or biofuel industries.

In terms of in vivo delivery, AAV is advantageous over other viralvectors for a couple of reasons:

-   -   Low toxicity (this may be due to the purification method not        requiring ultra centrifugation of cell particles that can        activate the immune response)    -   probability of causing insertional mutagenesis because it        doesn't integrate into the host genome.

AAV has a packaging limit of 4.5 or 4.75 Kb. This means that Cas9 aswell as a promoter and transcription terminator have to be all fit intothe same viral vector. Constructs larger than 4.5 or 4.75 Kb will leadto significantly reduced virus production. SpCas9 is quite large, thegene itself is over 4.1 Kb, which makes it difficult for packing intoAAV. Therefore embodiments of the invention include utilizing homologsof Cas9 that are shorter. For example:

Species Cas9 Size Corynebacter diphtheriae 3252 Eubacterium ventriosum3321 Streptococcus pasteurianus 3390 Lactobacillus farciminis 3378Sphaerochaeta globus 3537 Azospirillum B510 3504 Gluconacetobacterdiazotrophicus 3150 Neisseria cinerea 3246 Roseburia intestinalis 3420Parvibaculum lavamentivorans 3111 Staphylococcus aureus 3159Nitratifractor salsuginis DSM 16511 3396 Campylobacter lari CF89-12 3009Streptococcus thermophilus LMD-9 3396

These species are therefore, in general, preferred Cas9 species.Applicants have shown delivery and in vivo mouse brain Cas9 expressiondata.

Two ways to package Cas9 coding nucleic acid molecules, e.g., DNA, intoviral vectors to mediate genome modification in vivo are preferred:

To achieve NHEJ-mediated gene knockout:Single virus vector:

Vector containing two or more expression cassettes:

Promoter-Cas9 coding nucleic acid molecule-terminator

Prom oter-gRNA1-terminator

Promoter-gRNA2-terminator

Promoter-gRNA(N)-terminator (up to size limit of vector)

Double virus vector:

Vector 1 containing one expression cassette for driving the expressionof Cas9

Promoter-Cas9 coding nucleic acid molecule-terminator

Vector 2 containing one more expression cassettes for driving theexpression of one or

more guideRNAs

Promoter-gRNA1-terminator

Promoter-gRNA(N)-terminator (up to size limit of vector)

To mediate homology-directed repair.In addition to the single and double virus vector approaches describedabove, an additional vector is used to deliver a homology-direct repairtemplate.

Promoter used to drive Cas9 coding nucleic acid molecule expression caninclude:

-   -   AAV ITR can serve as a promoter: this is advantageous for        eliminating the need for an additional promoter element (which        can take up space in the vector). The additional space freed up        can be used to drive the expression of additional elements        (gRNA, etc). Also, ITR activity is relatively weaker, so can be        used to reduce toxicity due to over expression of Cas9.    -   For ubiquitous expression, can use promoters: CMV, CAG, CBh,        PGK, SV40, Ferritin heavy or light chains, etc.    -   For brain expression, can use promoters: Synapsinl for all        neurons, CaMKIIalpha for excitatory neurons, GAD67 or GAD65 or        VGAT for GABAergic neurons, etc.    -   For liver expression, can use Albumin promoter    -   For lung expression, can use SP-B    -   For endothelial cells, can use ICAM    -   For hematopoietic cells can use IFNbeta or CD45    -   For Osteoblasts can use OG-2    -   Promoter used to drive guide RNA can include:    -   Pol III promoters such as U6 or H1    -   Use of Pol II promoter and intronic cassettes to express gRNA.

As to AAV, the AAV can be AAV1, AAV2, AAV5 or any combination thereof.One can select the AAV of the AAV with regard to the cells to betargeted; e.g., one can select AAV serotypes 1, 2, 5 or a hybrid orcapsid AAV1, AAV2, AAV5 or any combination thereof for targeting brainor neuronal cells; and one can select AAV4 for targeting cardiac tissue.AAV8 is useful for delivery to the liver. The above promoters andvectors are preferred individually.

RNA delivery is also a useful method of in vivo delivery. FIG. 9 showsdelivery and in vivo mouse brain Cas9 expression data. It is possible todeliver Cas9 and gRNA (and, for instance, HR repair template) into cellsusing liposomes or nanoparticles. Thus delivery of the CRISPR enzyme,such as a Cas9 and/or delivery of the RNAs of the invention may be inRNA form and via microvesicles, liposomes or nanoparticles. For example,Cas9 mRNA and gRNA can be packaged into liposomal particles for deliveryin vivo. Liposomal transfection reagents such as Invivofectamine fromLife Technologies and other reagents on the market can effectivelydeliver RNA molecules into the liver.

Enhancing NHEJ or HR efficiency is also helpful for delivery. It ispreferred that NHEJ efficiency is enhanced by co-expressingend-processing enzymes such as Trex2 (Dumitrache et al. Genetics. 2011August; 188(4): 787-797). It is preferred that HR efficiency isincreased by transiently inhibiting NHEJ machineries such as Ku70 andKu86. HR efficiency can also be increased by co-expressing prokaryoticor eukaryotic homologous recombination enzymes such as RecBCD, RecA.

Various means of delivery are described herein, and further discussed inthis section.

Viral Delivery:

The CRISPR enzyme, for instance a Cas9, and/or any of the present RNAs,for instance a guide RNA, can be delivered using adeno associated virus(AAV), lentivirus, adenovirus or other viral vector types, orcombinations thereof. Cas9 and one or more guide RNAs can be packagedinto one or more viral vectors. In some embodiments, the viral vector isdelivered to the tissue of interest by, for example, an intramuscularinjection, while other times the viral delivery is via intravenous,transdermal, intranasal, oral, mucosal, or other delivery methods. Suchdelivery may be either via a single dose, or multiple doses. One skilledin the art understands that the actual dosage to be delivered herein mayvary greatly depending upon a variety of factors, such as the vectorchose, the target cell, organism, or tissue, the general condition ofthe subject to be treated, the degree of transformation/modificationsought, the administration route, the administration mode, the type oftransformation/modification sought, etc.

Such a dosage may further contain, for example, a carrier (water,saline, ethanol, glycerol, lactose, sucrose, calcium phosphate, gelatin,dextran, agar, pectin, peanut oil, sesame oil, etc.), a diluent, apharmaceutically-acceptable carrier (e.g., phosphate-buffered saline), apharmaceutically-acceptable excipient, an adjuvant to enhanceantigenicity, an immunostimulatory compound or molecule, and/or othercompounds known in the art. The adjuvant herein may contain a suspensionof minerals (alum, aluminum hydroxide, aluminum phosphate) on whichantigen is adsorbed; or water-in-oil emulsion in which antigen solutionis emulsified in oil (MF-59, Freund's incomplete adjuvant), sometimeswith the inclusion of killed mycobacteria (Freund's complete adjuvant)to further enhance antigenicity (inhibits degradation of antigen and/orcauses influx of macrophages). Adjuvants also include immunostimulatorymolecules, such as cytokines, costimulatory molecules, and for example,immunostimulatory DNA or RNA molecules, such as CpG oligonucleotides.Such a dosage formulation is readily ascertainable by one skilled in theart. The dosage may further contain one or more pharmaceuticallyacceptable salts such as, for example, a mineral acid salt such as ahydrochloride, a hydrobromide, a phosphate, a sulfate, etc.; and thesalts of organic acids such as acetates, propionates, malonates,benzoates, etc. Additionally, auxiliary substances, such as wetting oremulsifying agents, pH buffering substances, gels or gelling materials,flavorings, colorants, microspheres, polymers, suspension agents, etc.may also be present herein. In addition, one or more other conventionalpharmaceutical ingredients, such as preservatives, humectants,suspending agents, surfactants, antioxidants, anticaking agents,fillers, chelating agents, coating agents, chemical stabilizers, etc.may also be present, especially if the dosage form is a reconstitutableform. Suitable exemplary ingredients include microcrystalline cellulose,carboxymethylcellulose sodium, polysorbate 80, phenylethyl alcohol,chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propylgallate, the parabens, ethyl vanillin, glycerin, phenol,parachlorophenol, gelatin, albumin and a combination thereof. A thoroughdiscussion of pharmaceutically acceptable excipients is available inREMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991) which isincorporated by reference herein.

In an embodiment herein the delivery is via an adenovirus, which may beat a single booster dose containing at least 1×10⁵ particles (alsoreferred to as particle units, pu) of adenoviral vector. In anembodiment herein, the dose preferably is at least about 1×10⁶ particles(for example, about 1×10⁶-1×10¹² particles), more preferably at leastabout 1×10⁷ particles, more preferably at least about 1×10⁸ particles(e.g., about 1×10⁸-1×10¹¹ particles or about 1×10⁸-1×10¹² particles),and most preferably at least about 1×10° particles (e.g., about1*×10⁹-1×10¹⁰ particles or about 1×10⁹-1×10¹² particles), or even atleast about 1×10¹⁰ particles (e.g., about 1×10¹⁰-1×10¹² particles) ofthe adenoviral vector. Alternatively, the dose comprises no more thanabout 1×10¹⁴ particles, preferably no more than about 1×10¹³ particles,even more preferably no more than about 1×10¹² particles, even morepreferably no more than about 1×10¹¹ particles, and most preferably nomore than about 1×10¹⁰ particles (e.g., no more than about 1×10⁹articles). Thus, the dose may contain a single dose of adenoviral vectorwith, for example, about 1×10⁶ particle units (pu), about 2×10⁶ pu,about 4×10⁶ pu, about 1×10⁷ pu, about 2×10⁷ pu, about 4×10⁷ pu, about1×10⁸ pu, about 2×10⁸ pu, about 4×10⁸ pu, about 1×10⁹ pu, about 2×10⁹pu, about 4×10⁹ pu, about 1×10¹⁰ pu, about 2×10¹⁰ pu, about 4×10¹⁰ pu,about 1×10¹¹ pu, about 2×10¹¹ pu, about 4×10¹¹ pu, about 1×10¹² pu,about 2×10¹² pu, or about 4×10¹² pu of adenoviral vector. See, forexample, the adenoviral vectors in U.S. Pat. No. 8,454,972 B2 to Nabel,et. al., granted on Jun. 4, 2013; incorporated by reference herein, andthe dosages at col 29, lines 36-58 thereof. In an embodiment herein, theadenovirus is delivered via multiple doses.

In an embodiment herein, the delivery is via an AAV. A therapeuticallyeffective dosage for in vivo delivery of the AAV to a human is believedto be in the range of from about 20 to about 50 ml of saline solutioncontaining from about 1×10¹⁰ to about 1×10¹⁰ functional AAV/ml solution.The dosage may be adjusted to balance the therapeutic benefit againstany side effects. In an embodiment herein, the AAV dose is generally inthe range of concentrations of from about 1×10⁵ to 1×10⁵⁰ genomes AAV,from about 1×10⁸ to 1×10²⁰ genomes AAV, from about 1×10¹⁰ to about1×10¹⁶ genomes, or about 1×10¹¹ to about 1×10¹⁶ genomes AAV. A humandosage may be about 1×10¹³ genomes AAV. Such concentrations may bedelivered in from about 0.001 ml to about 100 ml, about 0.05 to about 50ml, or about 10 to about 25 ml of a carrier solution. Other effectivedosages can be readily established by one of ordinary skill in the artthrough routine trials establishing dose response curves. See, forexample, U.S. Pat. No. 8,404,658 B2 to Hajjar, et al., granted on Mar.26, 2013, at col. 27, lines 45-60.

In an embodiment herein the delivery is via a plasmid. In such plasmidcompositions, the dosage should be a sufficient amount of plasmid toelicit a response. For instance, suitable quantities of plasmid DNA inplasmid compositions can be from about 0.1 to about 2 mg, or from about1 μg to about 10 μg.

The doses herein are based on an average 70 kg individual. The frequencyof administration is within the ambit of the medical or veterinarypractitioner (e.g., physician, veterinarian), or scientist skilled inthe art.

Cas9 and one or more guide RNA can be delivered using adeno associatedvirus (AAV), lentivirus, adenovirus or other plasmid or viral vectortypes, using formulations and doses from, for example, U.S. Pat. No.8,454,972 (formulations, doses for adenovirus), U.S. Pat. No. 8,404,658(formulations, doses for AAV) and U.S. Pat. No. 5,846,946 (formulations,doses for DNA plasmids) and from clinical trials and publicationsregarding the clinical trials involving lentivirus, AAV and adenovirus.For examples, for AAV, the route of administration, formulation and dosecan be as in U.S. Pat. No. 8,454,972 and as in clinical trials involvingAAV. For Adenovirus, the route of administration, formulation and dosecan be as in U.S. Pat. No. 8,404,658 and as in clinical trials involvingadenovirus. For plasmid delivery, the route of administration,formulation and dose can be as in U.S. Pat. No. 5,846,946 and as inclinical studies involving plasmids. Doses may be based on orextrapolated to an average 70 kg individual, and can be adjusted forpatients, subjects, mammals of different weight and species. Frequencyof administration is within the ambit of the medical or veterinarypractitioner (e.g., physician, veterinarian), depending on usual factorsincluding the age, sex, general health, other conditions of the patientor subject and the particular condition or symptoms being addressed.

The viral vectors can be injected into the tissue of interest. Forcell-type specific genome modification, the expression of Cas9 can bedriven by a cell-type specific promoter. For example, liver-specificexpression might use the Albumin promoter and neuron-specific expressionmight use the Synapsin I promoter.

RNA Delivery:

The CRISPR enzyme, for instance a Cas9, and/or any of the present RNAs,for instance a guide RNA, can also be delivered in the form of RNA. Cas9mRNA can be generated using in vitro transcription. For example, Cas9mRNA can be synthesized using a PCR cassette containing the followingelements: T7promoter-kozak sequence (GCCACC)-Cas9-3′ UTR from betaglobin-polyA tail (a string of 120 or more adenines (SEQ ID NO: 350)).The cassette can be used for transcription by T7 polymerase. Guide RNAscan also be transcribed using in vitro transcription from a cassettecontaining T7 promoter-GG-guide RNA sequence.

To enhance expression and reduce toxicity, the CRISPR enzyme and/orguide RNA can be modified using pseudo-U or 5-Methyl-C.

CRISPR enzyme mRNA and guide RNA may be delivered simultaneously usingnanoparticles or lipid envelopes.

For example, Su X, Fricke J, Kavanagh D G, Irvine D J (“In vitro and invivo mRNA delivery using lipid-enveloped pH-responsive polymernanoparticles” Mol Pharm. 2011 Jun. 6; 8(3):774-87. doi:10.1021/mp100390w. Epub 2011 Apr. 1) describes biodegradable core-shellstructured nanoparticles with a poly(β-amino ester) (PBAE) coreenveloped by a phospholipid bilayer shell. These were developed for invivo mRNA delivery. The pH-responsive PBAE component was chosen topromote endosome disruption, while the lipid surface layer was selectedto minimize toxicity of the polycation core. Such are, therefore,preferred for delivering RNA of the present invention.

Furthermore, Michael S D Kormann et al. (“Expression of therapeuticproteins after delivery of chemically modified mRNA in mice: NatureBiotechnology, Volume:29, Pages: 154-157 (2011) Published online 9 Jan.2011) describes the use of lipid envelopes to deliver RNA. Use of lipidenvelopes is also preferred in the present invention.

mRNA delivery methods are especially promising for liver deliverycurrently.

CRISPR enzyme mRNA and guide RNA might also be delivered separately.CRISPR enzyme mRNA can be delivered prior to the guide RNA to give timefor CRISPR enzyme to be expressed. CRISPR enzyme mRNA might beadministered 1-12 hours (preferably around 2-6 hours) prior to theadministration of guide RNA.

Alternatively, CRISPR enzyme mRNA and guide RNA can be administeredtogether. Advantageously, a second booster dose of guide RNA can beadministered 1-12 hours (preferably around 2-6 hours) after the initialadministration of CRISPR enzyme mRNA+ guide RNA.

Additional administrations of CRISPR enzyme mRNA and/or guide RNA mightbe useful to achieve the most efficient levels of genome modification.

For minimization of toxicity and off-target effect, it will be importantto control the concentration of CRISPR enzyme mRNA and guide RNAdelivered. Optimal concentrations of CRISPR enzyme mRNA and guide RNAcan be determined by testing different concentrations in a cellular oranimal model and using deep sequencing the analyze the extent ofmodification at potential off-target genomic loci. For example, for theguide sequence targeting 5′-GAGTCCGAGCAGAAGAAGAA-3′ (SEQ ID NO: 2) inthe EMX1 gene of the human genome, deep sequencing can be used to assessthe level of modification at the following two off-target loci, 1:5′-GAGTCCTAGCAGGAGAAGAA-3′ (SEQ ID NO: 3) and 2:5′-GAGTCTAAGCAGAAGAAGAA-3′ (SEQ ID NO: 4). The concentration that givesthe highest level of on-target modification while minimizing the levelof off-target modification should be chosen for in vivo delivery.

Alternatively, to minimize the level of toxicity and off-target effect,CRISPR enzyme nickase mRNA (for example S. pyogenes Cas9 with either aD10A or a H840A mutation) can be delivered with a pair of guide RNAstargeting a site of interest. The two guide RNAs need to be spaced asfollows. Guide sequences in red (single underline) and blue (doubleunderline) respectively (these examples are based on the PAM requirementfor Streptococcus pyogenes Cas9.

Overhang length (bp)Guide RNA design (guide sequence and PAM color coded) 14

(SEQ ID NO: 5)3′-NNNNNNNNNNNNNNNNNNNNGGNNNNNNNNNNNNNNNNNNNNNNCCNNNNNNNNNNNNNN-5′(SEQ ID NO: 6) 13

(SEQ ID NO: 7)

(SEQ ID NO: 8) 12

(SEQ ID NO: 9)3′-NNNNNNNNNNNNNNNNNNNNGGNNNNNNNNNNNNNNNNNNNNNCCNNNNNNNNNNNNNNN-5′(SEQ ID NO: 10) 11

(SEQ ID NO: 11)

(SEQ ID NO: 12) 10

(SEQ ID NO: 13)3′-NNNNNNNNNNNNNNNNNNNNGGNNNNNNNNNNNNNNNNNNCCNNNNNNNNNNNNNNNNNN-5′(SEQ ID NO: 14) 9

(SEQ ID NO: 15)

(SEQ ID NO: 16) 8

(SEQ ID NO: 17)3′-NNNNNNNNNNNNNNNNNNNNGGNNNNNNNNNNNNNNNNCCNNNNNNNNNNNNNNNNNNNN-5′(SEQ ID NO: 18) 7

(SEQ ID NO: 19)

(SEQ ID NO: 20) 6

(SEQ ID NO: 21)

(SEQ ID NO: 22) 5

(SEQ ID NO: 23)

(SEQ ID NO: 24) 4

(SEQ ID NO: 25)

(SEQ ID NO: 26) 3

(SEQ ID NO: 27)

(SEQ ID NO: 28) 2

(SEQ ID NO: 29)

(SEQ ID NO: 30) 1

(SEQ ID NO: 31)

(SEQ ID NO: 32) blunt

(SEQ ID NO: 33)

(SEQ ID NO: 34) 1

(SEQ ID NO: 35)

(SEQ ID NO: 36) 2

(SEQ ID NO: 37)

(SEQ ID NO: 38) 3

(SEQ ID NO: 39)

(SEQ ID NO: 40) 4

(SEQ ID NO: 41)

(SEQ ID NO: 42) 5

(SEQ ID NO: 43)

(SEQ ID NO: 44) 6

(SEQ ID NO: 45)

(SEQ ID NO: 46) 7

(SEQ ID NO: 47)

(SEQ ID NO: 48) 8

(SEQ ID NO: 49)

(SEQ ID NO: 50) 12

(SEQ ID NO: 51)

(SEQ ID NO: 52) 13

(SEQ ID NO: 53)

(SEQ ID NO: 54) 14

(SEQ ID NO: 53)

(SEQ ID NO: 55) 15

(SEQ ID NO: 53)

(SEQ ID NO: 56) 16

(SEQ ID NO: 53)

(SEQ ID NO: 57) 17

(SEQ ID NO: 53)

(SEQ ID NO: 58)

Further interrogation of the system have given Applicants evidence ofthe 5′ overhang (see, e.g., Ran et al., Cell. 2013 Sep. 12;154(6):1380-9 and U.S. Provisional Patent Application Ser. No.61/871,301 filed Aug. 28, 2013). Applicants have further identifiedparameters that relate to efficient cleavage by the Cas9 nickase mutantwhen combined with two guide RNAs and these parameters include but arenot limited to the length of the 5′ overhang. In embodiments of theinvention the 5′ overhang is at most 200 base pairs, preferably at most100 base pairs, or more preferably at most 50 base pairs. In embodimentsof the invention the 5′ overhang is at least 26 base pairs, preferablyat least 30 base pairs or more preferably 34-50 base pairs or 1-34 basepairs. In other preferred methods of the invention the first guidesequence directing cleavage of one strand of the DNA duplex near thefirst target sequence and the second guide sequence directing cleavageof other strand near the second target sequence results in a blunt cutor a 3′ overhang. In embodiments of the invention the 3′ overhang is atmost 150, 100 or 25 base pairs or at least 15, 10 or 1 base pairs. Inpreferred embodiments the 3′ overhang is 1-100 basepairs.

Aspects of the invention relate to the expression of the gene productbeing decreased or a template polynucleotide being further introducedinto the DNA molecule encoding the gene product or an interveningsequence being excised precisely by allowing the two 5′ overhangs toreanneal and ligate or the activity or function of the gene productbeing altered or the expression of the gene product being increased. Inan embodiment of the invention, the gene product is a protein.

Only sgRNA pairs creating 5′ overhangs with less than 8 bp overlapbetween the guide sequences (offset greater than −8 bp) were able tomediate detectable indel formation. Importantly, each guide used inthese assays is able to efficiently induce indels when paired withwildtype Cas9, indicating that the relative positions of the guide pairsare the most important parameters in predicting double nicking activity.

Since Cas9n and Cas9H840A nick opposite strands of DNA, substitution ofCas9n with Cas9H840A with a given sgRNA pair should result in theinversion of the overhang type. For example, a pair of sgRNAs that willgenerate a 5′ overhang with Cas9n should in principle generate thecorresponding 3′ overhang instead. Therefore, sgRNA pairs that lead tothe generation of a 3′ overhang with Cas9n might be used with Cas9H840Ato generate a 5′ overhang. Unexpectedly, Applicants tested Cas9H840Awith a set of sgRNA pairs designed to generate both 5′ and 3′ overhangs(offset range from −278 to +58 bp), but were unable to observe indelformation. Further work may be needed to identify the necessary designrules for sgRNA pairing to allow double nicking by Cas9H840A.

Additional delivery options for the brain include encapsulation ofCRISPR enzyme and guide RNA in the form of either DNA or RNA intoliposomes and conjugating to molecular Trojan horses for trans-bloodbrain barrier (BBB) delivery. Molecular Trojan horses have been shown tobe effective for delivery of B-gal expression vectors into the brain ofnon-human primates. The same approach can be used to delivery vectorscontaining CRISPR enzyme and guide RNA. For instance, Xia C F and BoadoR J, Pardridge W M (“Antibody-mediated targeting of siRNA via the humaninsulin receptor using avidin-biotin technology.” Mol Pharm. 2009May-June; 6(3):747-51. doi: 10.1021/mp800194) describes how delivery ofshort interfering RNA (siRNA) to cells in culture, and in vivo, ispossible with combined use of a receptor-specific monoclonal antibody(mAb) and avidin-biotin technology. The authors also report that becausethe bond between the targeting mAb and the siRNA is stable withavidin-biotin technology, and RNAi effects at distant sites such asbrain are observed in vivo following an intravenous administration ofthe targeted siRNA.

Zhang Y, Schlachetzki F, Pardridge W M. (“Global non-viral gene transferto the primate brain following intravenous administration.” Mol Ther.2003 January; 7(1):11-8.) describe how expression plasmids encodingreporters such as luciferase were encapsulated in the interior of an“artificial virus” comprised of an 85 nm pegylated immunoliposome, whichwas targeted to the rhesus monkey brain in vivo with a monoclonalantibody (MAb) to the human insulin receptor (HIR). The HIRMAb enablesthe liposome carrying the exogenous gene to undergo transcytosis acrossthe blood-brain barrier and endocytosis across the neuronal plasmamembrane following intravenous injection. The level of luciferase geneexpression in the brain was 50-fold higher in the rhesus monkey ascompared to the rat. Widespread neuronal expression of thebeta-galactosidase gene in primate brain was demonstrated by bothhistochemistry and confocal microscopy. The authors indicate that thisapproach makes feasible reversible adult transgenics in 24 hours.Accordingly, the use of immunoliposome is preferred. These may be usedin conjunction with antibodies to target specific tissues or cellsurface proteins.

Other means of delivery or RNA are also preferred, such as viananoparticles (Cho, S., Goldberg, M., Son, S., Xu, Q., Yang, F., Mei,Y., Bogatyrev, S., Langer, R. and Anderson, D., Lipid-like nanoparticlesfor small interfering RNA delivery to endothelial cells, AdvancedFunctional Materials, 19: 3112-3118, 2010) or exosomes (Schroeder, A.,Levins, C., Cortez, C., Langer, R., and Anderson, D., Lipid-basednanotherapeutics for siRNA delivery, Journal of Internal Medicine, 267:9-21, 2010, PMID: 20059641). Indeed, exozomes have been shown to beparticularly useful in delivery siRNA, a system with some parallels tothe CRISPR system. For instance, El-Andaloussi S, et al.(“Exosome-mediated delivery of siRNA in vitro and in vivo.” Nat Protoc.2012 December; 7(12):2112-26. doi: 10.1038/nprot.2012.131. Epub 2012Nov. 15) describe how exosomes are promising tools for drug deliveryacross different biological barriers and can be harnessed for deliveryof siRNA in vitro and in vivo. Their approach is to generate targetedexosomes through transfection of an expression vector, comprising anexosomal protein fused with a peptide ligand. The exosomes are thenpurify and characterized from transfected cell supernatant, then siRNAis loaded into the exosomes.

Targeted deletion of genes is preferred. Examples are exemplified inExample 12. Preferred are, therefore, genes involved in cholesterolbiosynthesis, fatty acid biosynthesis, and other metabolic disorders,genes encoding mis-folded proteins involved in amyloid and otherdiseases, oncogenes leading to cellular transformation, latent viralgenes, and genes leading to dominant-negative disorders, amongst otherdisorders. As exemplified here, Applicants prefer gene delivery of aCRISPR-Cas system to the liver, brain, ocular, epithelial, hematopoetic,or another tissue of a subject or a patient in need thereof, sufferingfrom metabolic disorders, amyloidosis and protein-aggregation relateddiseases, cellular transformation arising from genetic mutations andtranslocations, dominant negative effects of gene mutations, latentviral infections, and other related symptoms, using either viral ornanoparticle delivery system.

Therapeutic applications of the CRISPR-Cas system include Glaucoma,Amyloidosis, and Huntington's disease. These are exemplified in Example14 and the features described therein are preferred alone or incombination.

As an example, chronic infection by HIV-1 may be treated or prevented.In order to accomplish this, one may generate CRISPR-Cas guide RNAs thattarget the vast majority of the HIV-1 genome while taking into accountHIV-1 strain variants for maximal coverage and effectiveness. One mayaccomplish delivery of the CRISPR-Cas system by conventional adenoviralor lentiviral-mediated infection of the host immune system. Depending onapproach, host immune cells could be a) isolated, transduced withCRISPR-Cas, selected, and re-introduced in to the host or b) transducedin vivo by systemic delivery of the CRISPR-Cas system. The firstapproach allows for generation of a resistant immune population whereasthe second is more likely to target latent viral reservoirs within thehost. This is discussed in more detail in the Examples section.

It is also envisaged that the present invention generates a geneknockout cell library. Each cell may have a single gene knocked out.This is exemplified in Example 17.

One may make a library of ES cells where each cell has a single geneknocked out, and the entire library of ES cells will have every singlegene knocked out. This library is useful for the screening of genefunction in cellular processes as well as diseases. To make this celllibrary, one may integrate Cas9 driven by an inducible promoter (e.g.doxycycline inducible promoter) into the ES cell. In addition, one mayintegrate a single guide RNA targeting a specific gene in the ES cell.To make the ES cell library, one may simply mix ES cells with a libraryof genes encoding guide RNAs targeting each gene in the human genome.One may first introduce a single BxB1 attB site into the AAVS1 locus ofthe human ES cell. Then one may use the BxB1 integrase to facilitate theintegration of individual guide RNA genes into the BxB1 attB site inAAVS1 locus. To facilitate integration, each guide RNA gene may becontained on a plasmid that carries of a single attP site. This way BxB1will recombine the attB site in the genome with the attP site on theguide RNA containing plasmid. To generate the cell library, one may takethe library of cells that have single guide RNAs integrated and induceCas9 expression. After induction, Cas9 mediates double strand break atsites specified by the guide RNA.

Chronic administration of protein therapeutics may elicit unacceptableimmune responses to the specific protein. The immunogenicity of proteindrugs can be ascribed to a few immunodominant helper T lymphocyte (HTL)epitopes. Reducing the MHC binding affinity of these HTL epitopescontained within these proteins can generate drugs with lowerimmunogenicity (Tangri S, et al. (“Rationally engineered therapeuticproteins with reduced immunogenicity” J Immunol. 2005 Mar. 15;174(6):3187-96.) In the present invention, the immunogenicity of theCRISPR enzyme in particular may be reduced following the approach firstset out in Tangri et al with respect to erythropoietin and subsequentlydeveloped. Accordingly, directed evolution or rational design may beused to reduce the immunogenicity of the CRISPR enzyme (for instance aCas9) in the host species (human or other species).

In Example 28, Applicants used 3 guideRNAs of interest and able tovisualize efficient DNA cleavage in vivo occurring only in a smallsubset of cells. Essentially, what Applicants have shown here istargeted in vivo cleavage. In particular, this provides proof of conceptthat specific targeting in higher organisms such as mammals can also beachieved. It also highlights multiplex aspect in that multiple guidesequences (i.e. separate targets) can be used simultaneously (in thesense of co-delivery). In other words, Applicants used a multipleapproach, with several different sequences targeted at the same time,but independently.

A suitable example of a protocol for producing AAV, a preferred vectorof the invention is provided in Example 29.

Trinucleotide repeat disorders are preferred conditions to be treated.These are also exemplified herein.

According to another aspect, a method of gene therapy for the treatmentof a subject having a mutation in the CFTR gene is provided andcomprises administering a therapeutically effective amount of aCRISPR-Cas gene therapy particle, optionally via a biocompatiblepharmaceutical carrier, to the cells of a subject. Preferably, thetarget DNA comprises the mutation deltaF508. In general, it is ofpreferred that the mutation is repaired to the wildtype. In this case,the mutation is a deletion of the three nucleotides that comprise thecodon for phenylalanine (F) at position 508. Accordingly, repair in thisinstance requires reintroduction of the missing codon into the mutant.

To implement this Gene Repair Strategy, it is preferred that anadenovirus/AAV vector system is introduced into the host cell, cells orpatient. Preferably, the system comprises a Cas9 (or Cas9 nickase) andthe guide RNA along with a adenovirus/AAV vector system comprising thehomology repair template containing the F508 residue. This may beintroduced into the subject via one of the methods of delivery discussedearlier. The CRISPR-Cas system may be guided by the CFTRdelta 508chimeric guide RNA. It targets a specific site of the CFTR genomic locusto be nicked or cleaved. After cleavage, the repair template is insertedinto the cleavage site via homologous recombination correcting thedeletion that results in cystic fibrosis or causes cystic fibrosisrelated symptoms. This strategy to direct delivery and provide systemicintroduction of CRISPR systems with appropriate guide RNAs can beemployed to target genetic mutations to edit or otherwise manipulategenes that cause metabolic, liver, kidney and protein diseases anddisorders such as those in Table B.

For an example of CFTRdelta508 chimeric guide RNA, see Example whichdemonstrates gene transfer or gene delivery of a CRISPR-Cas system inairways of subject or a patient in need thereof, suffering from cysticfibrosis or from cystic fibrosis (CF) related symptoms, usingadeno-associated virus (AAV) particles. In particular, they exemplify arepair strategy for Cystic Fibrosis delta F508 mutation. This type ofstrategy should apply across all organisms. With particular reference toCF, suitable patients may include: Human, non-primate human, canine,feline, bovine, equine and other domestic animals. In this instance,Applicants utilized a CRISPR-Cas system comprising a Cas9 enzyme totarget deltaF508 or other CFTR-inducing mutations.

The treated subjects in this instance receive pharmaceutically effectiveamount of aerosolized AAV vector system per lung endobronchiallydelivered while spontaneously breathing. As such, aerosolized deliveryis preferred for AAV delivery in general. An adenovirus or an AAVparticle may be used for delivery. Suitable gene constructs, eachoperably linked to one or more regulatory sequences, may be cloned intothe delivery vector. In this instance, the following constructs areprovided as examples: Cbh or EF1a promoter for Cas9, U6 or H1 promoterfor chimeric guide RNA): A preferred arrangement is to use aCFTRdelta508 targeting chimeric guide, a repair template for deltaF508mutation and a codon optimized Cas9 enzyme (preferred Cas9s are thosewith nuclease or nickase activity) with optionally one or more nuclearlocalization signal or sequence(s) (NLS(s)), e.g., two (2) NLSs.Constructs without NLS are also envisaged.

In order to identify the Cas9 target site, Applicants analyzed the humanCFTR genomic locus and identified the Cas9 target site. Preferably, ingeneral and in this CF case, the PAM may contain a NGG or a NNAGAAWmotif

Accordingly, in the case of CF, the present method comprisesmanipulation of a target sequence in a genomic locus of interestcomprising

delivering a non-naturally occurring or engineered compositioncomprising a viral vector system comprising one or more viral vectorsoperably encoding a composition for expression thereof, wherein thecomposition comprises:

a non-naturally occurring or engineered composition comprising a vectorsystem comprising one or more vectors comprising

I. a first regulatory element operably linked to a CRISPR-Cas systemchimeric RNA (chiRNA) polynucleotide sequence, wherein thepolynucleotide sequence comprises

(a) a guide sequence capable of hybridizing to the CF target sequence ina suitable mammalian cell,

(b) a tracr mate sequence, and

(c) a tracr sequence, and

II. a second regulatory element operably linked to an enzyme-codingsequence encoding a CRISPR enzyme comprising at least one or morenuclear localization sequences,

wherein (a), (b) and (c) are arranged in a 5′ to 3′ orientation,

wherein components I and II are located on the same or different vectorsof the system,

wherein when transcribed, the tracr mate sequence hybridizes to thetracr sequence and the guide sequence directs sequence-specific bindingof a CRISPR complex to the target sequence, and

wherein the CRISPR complex comprises the CRISPR enzyme complexed with(1) the guide sequence that is hybridizable to the target sequence, and(2) the tracr mate sequence that is hybridized to the tracr sequence. Inrespect of CF, preferred target DNA sequences comprise the CFTRdelta508mutation. A preferred PAM is described above. A preferred CRISPR enzymeis any Cas (described herein, but particularly that described in Example16).

Alternatives to CF include any genetic disorder and examples of theseare well known. Another preferred method or use of the invention is forcorrecting defects in the EMP2A and EMP2B genes that have beenidentified to be associated with Lafora disease.

In some embodiments, a “guide sequence” may be distinct from “guideRNA”. A guide sequence may refer to an approx. 20 bp sequence, withinthe guide RNA, that specifies the target site.

In some embodiments, the Cas9 is (or is derived from) the SpCas9. Insuch embodiments, preferred mutations are at any or all or positions 10,762, 840, 854, 863 and/or 986 of SpCas9 or corresponding positions inother Cas9s (which may be ascertained for instance by standard sequencecomparison tools. In particular, any or all of the following mutationsare preferred in SpCas9: D10A, E762A, H840A, N854A, N863A and/or D986A;as well as conservative substitution for any of the replacement aminoacids is also envisaged. The same (or conservative substitutions ofthese mutations) at corresponding positions in other Cas9s are alsopreferred. Particularly preferred are D10 and H840 in SpCas9. However,in other Cas9s, residues corresponding to SpCas9 D10 and H840 are alsopreferred. These are advantageous as they provide nickase activity.

It will be readily apparent that a host of other diseases can be treatedin a similar fashion. Some examples of genetic diseases caused bymutations are provided herein, but many more are known. The abovestrategy can be applied to these diseases.

The invention uses nucleic acids to bind target DNA sequences. This isadvantageous as nucleic acids are much easier and cheaper to produce andthe specificity can be varied according to the length of the stretchwhere homology is sought. Complex 3-D positioning of multiple fingers,for example is not required.

The terms “polynucleotide”, “nucleotide”, “nucleotide sequence”,“nucleic acid” and “oligonucleotide” are used interchangeably. Theyrefer to a polymeric form of nucleotides of any length, eitherdeoxyribonucleotides or ribonucleotides, or analogs thereof.Polynucleotides may have any three dimensional structure, and mayperform any function, known or unknown. The following are non-limitingexamples of polynucleotides: coding or non-coding regions of a gene orgene fragment, loci (locus) defined from linkage analysis, exons,introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, shortinterfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA),ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides,plasmids, vectors, isolated DNA of any sequence, isolated RNA of anysequence, nucleic acid probes, and primers. The term also encompassesnucleic-acid-like structures with synthetic backbones, see, e.g.,Eckstein, 1991; Baserga et al., 1992; Milligan, 1993; WO 97/03211; WO96/39154; Mata, 1997; Strauss-Soukup, 1997; and Samstag, 1996. Apolynucleotide may comprise one or more modified nucleotides, such asmethylated nucleotides and nucleotide analogs. If present, modificationsto the nucleotide structure may be imparted before or after assembly ofthe polymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter polymerization, such as by conjugation with a labeling component.

As used herein the term “wild type” is a term of the art understood byskilled persons and means the typical form of an organism, strain, geneor characteristic as it occurs in nature as distinguished from mutant orvariant forms.

As used herein the term “variant” should be taken to mean the exhibitionof qualities that have a pattern that deviates from what occurs innature.

The terms “non-naturally occurring” or “engineered” are usedinterchangeably and indicate the involvement of the hand of man. Theterms, when referring to nucleic acid molecules or polypeptides meanthat the nucleic acid molecule or the polypeptide is at leastsubstantially free from at least one other component with which they arenaturally associated in nature and as found in nature.

“Complementarity” refers to the ability of a nucleic acid to formhydrogen bond(s) with another nucleic acid sequence by eithertraditional Watson-Crick base pairing or other non-traditional types. Apercent complementarity indicates the percentage of residues in anucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crickbase pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9,10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary).“Perfectly complementary” means that all the contiguous residues of anucleic acid sequence will hydrogen bond with the same number ofcontiguous residues in a second nucleic acid sequence. “Substantiallycomplementary” as used herein refers to a degree of complementarity thatis at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refersto two nucleic acids that hybridize under stringent conditions.

As used herein, “stringent conditions” for hybridization refer toconditions under which a nucleic acid having complementarity to a targetsequence predominantly hybridizes with the target sequence, andsubstantially does not hybridize to non-target sequences. Stringentconditions are generally sequence-dependent, and vary depending on anumber of factors. In general, the longer the sequence, the higher thetemperature at which the sequence specifically hybridizes to its targetsequence. Non-limiting examples of stringent conditions are described indetail in Tijssen (1993), Laboratory Techniques In Biochemistry AndMolecular Biology-Hybridization With Nucleic Acid Probes Part I, SecondChapter “Overview of principles of hybridization and the strategy ofnucleic acid probe assay”, Elsevier, N.Y. Where reference is made to apolynucleotide sequence, then complementary or partially complementarysequences are also envisaged. These are preferably capable ofhybridising to the reference sequence under highly stringent conditions.Generally, in order to maximize the hybridization rate, relativelylow-stringency hybridization conditions are selected: about 20 to 25° C.lower than the thermal melting point (T_(m)). The T_(m) is thetemperature at which 50% of specific target sequence hybridizes to aperfectly complementary probe in solution at a defined ionic strengthand pH. Generally, in order to require at least about 85% nucleotidecomplementarity of hybridized sequences, highly stringent washingconditions are selected to be about 5 to 15° C. lower than the T_(m). Inorder to require at least about 70% nucleotide complementarity ofhybridized sequences, moderately-stringent washing conditions areselected to be about 15 to 30° C. lower than the T_(m). Highlypermissive (very low stringency) washing conditions may be as low as 50°C. below the T_(m), allowing a high level of mis-matching betweenhybridized sequences. Those skilled in the art will recognize that otherphysical and chemical parameters in the hybridization and wash stagescan also be altered to affect the outcome of a detectable hybridizationsignal from a specific level of homology between target and probesequences. Preferred highly stringent conditions comprise incubation in50% formamide, 5×SSC, and 1% SDS at 42° C., or incubation in 5×SSC and1% SDS at 65° C., with wash in 0.2×SSC and 0.1% SDS at 65° C.

“Hybridization” refers to a reaction in which one or morepolynucleotides react to form a complex that is stabilized via hydrogenbonding between the bases of the nucleotide residues. The hydrogenbonding may occur by Watson Crick base pairing, Hoogstein binding, or inany other sequence specific manner. The complex may comprise two strandsforming a duplex structure, three or more strands forming a multistranded complex, a single self-hybridizing strand, or any combinationof these. A hybridization reaction may constitute a step in a moreextensive process, such as the initiation of PCR, or the cleavage of apolynucleotide by an enzyme. A sequence capable of hybridizing with agiven sequence is referred to as the “complement” of the given sequence.

As used herein, the term “genomic locus” or “locus” (plural loci) is thespecific location of a gene or DNA sequence on a chromosome. A “gene”refers to stretches of DNA or RNA that encode a polypeptide or an RNAchain that has functional role to play in an organism and hence is themolecular unit of heredity in living organisms. For the purpose of thisinvention it may be considered that genes include regions which regulatethe production of the gene product, whether or not such regulatorysequences are adjacent to coding and/or transcribed sequences.Accordingly, a gene includes, but is not necessarily limited to,promoter sequences, terminators, translational regulatory sequences suchas ribosome binding sites and internal ribosome entry sites, enhancers,silencers, insulators, boundary elements, replication origins, matrixattachment sites and locus control regions.

As used herein, “expression of a genomic locus” or “gene expression” isthe process by which information from a gene is used in the synthesis ofa functional gene product. The products of gene expression are oftenproteins, but in non-protein coding genes such as rRNA genes or tRNAgenes, the product is functional RNA. The process of gene expression isused by all known life—eukaryotes (including multicellular organisms),prokaryotes (bacteria and archaea) and viruses to generate functionalproducts to survive. As used herein “expression” of a gene or nucleicacid encompasses not only cellular gene expression, but also thetranscription and translation of nucleic acid(s) in cloning systems andin any other context. As used herein, “expression” also refers to theprocess by which a polynucleotide is transcribed from a DNA template(such as into and mRNA or other RNA transcript) and/or the process bywhich a transcribed mRNA is subsequently translated into peptides,polypeptides, or proteins. Transcripts and encoded polypeptides may becollectively referred to as “gene product.” If the polynucleotide isderived from genomic DNA, expression may include splicing of the mRNA ina eukaryotic cell.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to polymers of amino acids of anylength. The polymer may be linear or branched, it may comprise modifiedamino acids, and it may be interrupted by non amino acids. The termsalso encompass an amino acid polymer that has been modified; forexample, disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, or any other manipulation, such asconjugation with a labeling component. As used herein the term “aminoacid” includes natural and/or unnatural or synthetic amino acids,including glycine and both the D or L optical isomers, and amino acidanalogs and peptidomimetics.

As used herein, the term “domain” or “protein domain” refers to a partof a protein sequence that may exist and function independently of therest of the protein chain.

As described in aspects of the invention, sequence identity is relatedto sequence homology. Homology comparisons may be conducted by eye, ormore usually, with the aid of readily available sequence comparisonprograms. These commercially available computer programs may calculatepercent (%) homology between two or more sequences and may alsocalculate the sequence identity shared by two or more amino acid ornucleic acid sequences. In some preferred embodiments, the cappingregion of the dTALEs described herein have sequences that are at least95% identical or share identity to the capping region amino acidsequences provided herein.

Sequence homologies may be generated by any of a number of computerprograms known in the art, for example BLAST or FASTA, etc. A suitablecomputer program for carrying out such an alignment is the GCG WisconsinBestfit package (University of Wisconsin, U.S.A; Devereux et al., 1984,Nucleic Acids Research 12:387). Examples of other software than mayperform sequence comparisons include, but are not limited to, the BLASTpackage (see Ausubel et al., 1999 ibid—Chapter 18), FASTA (Atschul etal., 1990, J. Mol. Biol., 403-410) and the GENEWORKS suite of comparisontools. Both BLAST and FASTA are available for offline and onlinesearching (see Ausubel et al., 1999 ibid, pages 7-58 to 7-60). Howeverit is preferred to use the GCG Bestfit program.

Percentage (%) sequence homology may be calculated over contiguoussequences, i.e., one sequence is aligned with the other sequence andeach amino acid or nucleotide in one sequence is directly compared withthe corresponding amino acid or nucleotide in the other sequence, oneresidue at a time. This is called an “ungapped” alignment. Typically,such ungapped alignments are performed only over a relatively shortnumber of residues.

Although this is a very simple and consistent method, it fails to takeinto consideration that, for example, in an otherwise identical pair ofsequences, one insertion or deletion may cause the following amino acidresidues to be put out of alignment, thus potentially resulting in alarge reduction in % homology when a global alignment is performed.Consequently, most sequence comparison methods are designed to produceoptimal alignments that take into consideration possible insertions anddeletions without unduly penalizing the overall homology or identityscore. This is achieved by inserting “gaps” in the sequence alignment totry to maximize local homology or identity.

However, these more complex methods assign “gap penalties” to each gapthat occurs in the alignment so that, for the same number of identicalamino acids, a sequence alignment with as few gaps aspossible—reflecting higher relatedness between the two comparedsequences—may achieve a higher score than one with many gaps. “Affinitygap costs” are typically used that charge a relatively high cost for theexistence of a gap and a smaller penalty for each subsequent residue inthe gap. This is the most commonly used gap scoring system. High gappenalties may, of course, produce optimized alignments with fewer gaps.Most alignment programs allow the gap penalties to be modified. However,it is preferred to use the default values when using such software forsequence comparisons. For example, when using the GCG Wisconsin Bestfitpackage the default gap penalty for amino acid sequences is −12 for agap and −4 for each extension.

Calculation of maximum % homology therefore first requires theproduction of an optimal alignment, taking into consideration gappenalties. A suitable computer program for carrying out such analignment is the GCG Wisconsin Bestfit package (Devereux et al., 1984Nuc. Acids Research 12 p387). Examples of other software than mayperform sequence comparisons include, but are not limited to, the BLASTpackage (see Ausubel et al., 1999 Short Protocols in Molecular Biology,4^(th) Ed. —Chapter 18), FASTA (Altschul et al., 1990 J. Mol. Biol.403-410) and the GENEWORKS suite of comparison tools. Both BLAST andFASTA are available for offline and online searching (see Ausubel etal., 1999, Short Protocols in Molecular Biology, pages 7-58 to 7-60).However, for some applications, it is preferred to use the GCG Bestfitprogram. A new tool, called BLAST 2 Sequences is also available forcomparing protein and nucleotide sequences (see FEMS Microbiol Lett.1999 174(2): 247-50; FEMS Microbiol Lett. 1999 177(1): 187-8 and thewebsite of the National Center for Biotechnology information at thewebsite of the National Institutes for Health).

Although the final % homology may be measured in terms of identity, thealignment process itself is typically not based on an all-or-nothingpair comparison. Instead, a scaled similarity score matrix is generallyused that assigns scores to each pair-wise comparison based on chemicalsimilarity or evolutionary distance. An example of such a matrixcommonly used is the BLOSUM62 matrix—the default matrix for the BLASTsuite of programs. GCG Wisconsin programs generally use either thepublic default values or a custom symbol comparison table, if supplied(see user manual for further details). For some applications, it ispreferred to use the public default values for the GCG package, or inthe case of other software, the default matrix, such as BLOSUM62.

Alternatively, percentage homologies may be calculated using themultiple alignment feature in DNASIS™ (Hitachi Software), based on analgorithm, analogous to CLUSTAL (Higgins D G & Sharp P M (1988), Gene73(1), 237-244). Once the software has produced an optimal alignment, itis possible to calculate % homology, preferably % sequence identity. Thesoftware typically does this as part of the sequence comparison andgenerates a numerical result.

The sequences may also have deletions, insertions or substitutions ofamino acid residues which produce a silent change and result in afunctionally equivalent substance. Deliberate amino acid substitutionsmay be made on the basis of similarity in amino acid properties (such aspolarity, charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues) and it is therefore useful to groupamino acids together in functional groups. Amino acids may be groupedtogether based on the properties of their side chains alone. However, itis more useful to include mutation data as well. The sets of amino acidsthus derived are likely to be conserved for structural reasons. Thesesets may be described in the form of a Venn diagram (Livingstone C. D.and Barton G. J. (1993) “Protein sequence alignments: a strategy for thehierarchical analysis of residue conservation” Comput. Appl. Biosci. 9:745-756) (Taylor W. R. (1986) “The classification of amino acidconservation” J. Theor. Biol. 119; 205-218). Conservative substitutionsmay be made, for example according to the table below which describes agenerally accepted Venn diagram grouping of amino acids.

Set Sub-set Hydrophobic F W Y H K M I L V A G C Aromatic F W Y HAliphatic I L V Polar W Y H K R E D C S T N Q Charged H K R E DPositively H K R charged Negatively E D charged Small V C A G S P T N DTiny A G S

Embodiments of the invention include sequences (both polynucleotide orpolypeptide) which may comprise homologous substitution (substitutionand replacement are both used herein to mean the interchange of anexisting amino acid residue or nucleotide, with an alternative residueor nucleotide) that may occur i.e., like-for-like substitution in thecase of amino acids such as basic for basic, acidic for acidic, polarfor polar, etc. Non-homologous substitution may also occur i.e., fromone class of residue to another or alternatively involving the inclusionof unnatural amino acids such as ornithine (hereinafter referred to asZ), diaminobutyric acid ornithine (hereinafter referred to as B),norleucine ornithine (hereinafter referred to as O), pyriylalanine,thienylalanine, naphthylalanine and phenylglycine.

Variant amino acid sequences may include suitable spacer groups that maybe inserted between any two amino acid residues of the sequenceincluding alkyl groups such as methyl, ethyl or propyl groups inaddition to amino acid spacers such as glycine or β-alanine residues. Afurther form of variation, which involves the presence of one or moreamino acid residues in peptoid form, may be well understood by thoseskilled in the art. For the avoidance of doubt, “the peptoid form” isused to refer to variant amino acid residues wherein the α-carbonsubstituent group is on the residue's nitrogen atom rather than theα-carbon. Processes for preparing peptides in the peptoid form are knownin the art, for example Simon R J et al., PNAS (1992) 89(20), 9367-9371and Horwell D C, Trends Biotechnol. (1995) 13(4), 132-134.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of immunology, biochemistry,chemistry, molecular biology, microbiology, cell biology, genomics andrecombinant DNA, which are within the skill of the art. See Sambrook,Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2ndedition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel,et al. eds., (1987)); the series METHODS IN ENZYMOLOGY (Academic Press,Inc.): PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, ALABORATORY MANUAL, and ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987)).

In one aspect, the invention provides for vectors that are used in theengineering and optimization of CRISPR-Cas systems.

A used herein, a “vector” is a tool that allows or facilitates thetransfer of an entity from one environment to another. It is a replicon,such as a plasmid, phage, or cosmid, into which another DNA segment maybe inserted so as to bring about the replication of the insertedsegment. Generally, a vector is capable of replication when associatedwith the proper control elements. In general, the term “vector” refersto a nucleic acid molecule capable of transporting another nucleic acidto which it has been linked. Vectors include, but are not limited to,nucleic acid molecules that are single-stranded, double-stranded, orpartially double-stranded; nucleic acid molecules that comprise one ormore free ends, no free ends (e.g. circular); nucleic acid moleculesthat comprise DNA, RNA, or both; and other varieties of polynucleotidesknown in the art. One type of vector is a “plasmid,” which refers to acircular double stranded DNA loop into which additional DNA segments canbe inserted, such as by standard molecular cloning techniques. Anothertype of vector is a viral vector, wherein virally-derived DNA or RNAsequences are present in the vector for packaging into a virus (e.g.retroviruses, replication defective retroviruses, adenoviruses,replication defective adenoviruses, and adeno-associated viruses(AAVs)). Viral vectors also include polynucleotides carried by a virusfor transfection into a host cell. Certain vectors are capable ofautonomous replication in a host cell into which they are introduced(e.g. bacterial vectors having a bacterial origin of replication andepisomal mammalian vectors). Other vectors (e.g., non-episomal mammalianvectors) are integrated into the genome of a host cell upon introductioninto the host cell, and thereby are replicated along with the hostgenome. Moreover, certain vectors are capable of directing theexpression of genes to which they are operatively-linked. Such vectorsare referred to herein as “expression vectors.” Common expressionvectors of utility in recombinant DNA techniques are often in the formof plasmids.

Recombinant expression vectors can comprise a nucleic acid of theinvention in a form suitable for expression of the nucleic acid in ahost cell, which means that the recombinant expression vectors includeone or more regulatory elements, which may be selected on the basis ofthe host cells to be used for expression, that is operatively-linked tothe nucleic acid sequence to be expressed. Within a recombinantexpression vector, “operably linked” is intended to mean that thenucleotide sequence of interest is linked to the regulatory element(s)in a manner that allows for expression of the nucleotide sequence (e.g.in an in vitro transcription/translation system or in a host cell whenthe vector is introduced into the host cell). With regards torecombination and cloning methods, mention is made of U.S. patentapplication Ser. No. 10/815,730, published Sep. 2, 2004 as US2004-0171156 A1, the contents of which are herein incorporated byreference in their entirety.

Aspects of the invention relate to bicistronic vectors for chimeric RNAand Cas9. Bicistronic expression vectors for chimeric RNA and Cas9 arepreferred. In general and particularly in this embodiment Cas9 ispreferably driven by the CBh promoter. The chimeric RNA may preferablybe driven by a U6 promoter. Ideally the two are combined. The chimericguide RNA typically consists of a 20 bp guide sequence (Ns) and this maybe joined to the tracr sequence (running from the first “U” of the lowerstrand to the end of the transcript). The tracr sequence may betruncated at various positions as indicated. The guide and tracrsequences are separated by the tracr-mate sequence, which may beGUUUUAGAGCUA (SEQ ID NO: 59). This may be followed by the loop sequenceGAAA as shown. Both of these are preferred examples. Applicants havedemonstrated Cas9-mediated indels at the human EMX1 and PVALB loci bySURVEYOR assays. ChiRNAs are indicated by their “+n” designation, andcrRNA refers to a hybrid RNA where guide and tracr sequences areexpressed as separate transcripts. Throughout this application, chimericRNA may also be called single guide, or synthetic guide RNA (sgRNA). Theloop is preferably GAAA, but it is not limited to this sequence orindeed to being only 4 bp in length. Indeed, preferred loop formingsequences for use in hairpin structures are four nucleotides in length,and most preferably have the sequence GAAA. However, longer or shorterloop sequences may be used, as may alternative sequences. The sequencespreferably include a nucleotide triplet (for example, AAA), and anadditional nucleotide (for example C or G). Examples of loop formingsequences include CAAA and AAAG.

The term “regulatory element” is intended to include promoters,enhancers, internal ribosomal entry sites (IRES), and other expressioncontrol elements (e.g. transcription termination signals, such aspolyadenylation signals and poly-U sequences). Such regulatory elementsare described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY:METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif (1990).Regulatory elements include those that direct constitutive expression ofa nucleotide sequence in many types of host cell and those that directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). A tissue-specific promoter maydirect expression primarily in a desired tissue of interest, such asmuscle, neuron, bone, skin, blood, specific organs (e.g. liver,pancreas), or particular cell types (e.g. lymphocytes). Regulatoryelements may also direct expression in a temporal-dependent manner, suchas in a cell-cycle dependent or developmental stage-dependent manner,which may or may not also be tissue or cell-type specific. In someembodiments, a vector comprises one or more pol III promoter (e.g. 1, 2,3, 4, 5, or more pol I promoters), one or more pol II promoters (e.g. 1,2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g.1, 2, 3, 4, 5, or more pol I promoters), or combinations thereof.Examples of pol III promoters include, but are not limited to, U6 and H1promoters. Examples of pol II promoters include, but are not limited to,the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally withthe RSV enhancer), the cytomegalovirus (CMV) promoter (optionally withthe CMV enhancer) [see, e.g., Boshart et al, Cell, 41:521-530 (1985)],the SV40 promoter, the dihydrofolate reductase promoter, the β-actinpromoter, the phosphoglycerol kinase (PGK) promoter, and the EF1apromoter. Also encompassed by the term “regulatory element” are enhancerelements, such as WPRE; CMV enhancers; the R-U5′ segment in LTR ofHTLV-I (Mol. Cell. Biol., Vol. 8(1), p. 466-472, 1988); SV40 enhancer;and the intron sequence between exons 2 and 3 of rabbit β-globin (Proc.Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981). It will beappreciated by those skilled in the art that the design of theexpression vector can depend on such factors as the choice of the hostcell to be transformed, the level of expression desired, etc. A vectorcan be introduced into host cells to thereby produce transcripts,proteins, or peptides, including fusion proteins or peptides, encoded bynucleic acids as described herein (e.g., clustered regularlyinterspersed short palindromic repeats (CRISPR) transcripts, proteins,enzymes, mutant forms thereof, fusion proteins thereof, etc.). Withregards to regulatory sequences, mention is made of U.S. patentapplication Ser. No. 10/491,026, the contents of which are incorporatedby reference herein in their entirety. With regards to promoters,mention is made of PCT publication WO 2011/028929 and U.S. applicationSer. No. 12/511,940, the contents of which are incorporated by referenceherein in their entirety.

Vectors can be designed for expression of CRISPR transcripts (e.g.nucleic acid transcripts, proteins, or enzymes) in prokaryotic oreukaryotic cells. For example, CRISPR transcripts can be expressed inbacterial cells such as Escherichia coli, insect cells (usingbaculovirus expression vectors), yeast cells, or mammalian cells.Suitable host cells are discussed further in Goeddel, GENE EXPRESSIONTECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif.(1990). Alternatively, the recombinant expression vector can betranscribed and translated in vitro, for example using T7 promoterregulatory sequences and T7 polymerase.

Vectors may be introduced and propagated in a prokaryote or prokaryoticcell. In some embodiments, a prokaryote is used to amplify copies of avector to be introduced into a eukaryotic cell or as an intermediatevector in the production of a vector to be introduced into a eukaryoticcell (e.g. amplifying a plasmid as part of a viral vector packagingsystem). In some embodiments, a prokaryote is used to amplify copies ofa vector and express one or more nucleic acids, such as to provide asource of one or more proteins for delivery to a host cell or hostorganism. Expression of proteins in prokaryotes is most often carriedout in Escherichia coli with vectors containing constitutive orinducible promoters directing the expression of either fusion ornon-fusion proteins. Fusion vectors add a number of amino acids to aprotein encoded therein, such as to the amino terminus of therecombinant protein. Such fusion vectors may serve one or more purposes,such as: (i) to increase expression of recombinant protein; (ii) toincrease the solubility of the recombinant protein; and (iii) to aid inthe purification of the recombinant protein by acting as a ligand inaffinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the fusionmoiety and the recombinant protein to enable separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Such enzymes, and their cognate recognitionsequences, include Factor Xa, thrombin and enterokinase. Example fusionexpression vectors include pGEX (Pharmacia Biotech Inc; Smith andJohnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly,Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathioneS-transferase (GST), maltose E binding protein, or protein A,respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 11d(Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185,Academic Press, San Diego, Calif (1990) 60-89).

In some embodiments, a vector is a yeast expression vector. Examples ofvectors for expression in yeast Saccharomyces cerivisae include pYepSecl(Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kuijan andHerskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al., 1987. Gene54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), andpicZ (InVitrogen Corp, San Diego, Calif.).

In some embodiments, a vector drives protein expression in insect cellsusing baculovirus expression vectors. Baculovirus vectors available forexpression of proteins in cultured insect cells (e.g., SF9 cells)include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3:2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170:31-39).

In some embodiments, a vector is capable of driving expression of one ormore sequences in mammalian cells using a mammalian expression vector.Examples of mammalian expression vectors include pCDM8 (Seed, 1987.Nature 329: 840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195).When used in mammalian cells, the expression vector's control functionsare typically provided by one or more regulatory elements. For example,commonly used promoters are derived from polyoma, adenovirus 2,cytomegalovirus, simian virus 40, and others disclosed herein and knownin the art. For other suitable expression systems for both prokaryoticand eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al.,MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

In some embodiments, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert, et al.,1987. Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame andEaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of Tcell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) andimmunoglobulins (Baneiji, et al., 1983. Cell 33: 729-740; Queen andBaltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci.USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al., 1985.Science 230: 912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990.Science 249: 374-379) and the α-fetoprotein promoter (Campes andTilghman, 1989. Genes Dev. 3: 537-546). With regards to theseprokaryotic and eukaryotic vectors, mention is made of U.S. Pat. No.6,750,059, the contents of which are incorporated by reference herein intheir entirety. Other embodiments of the invention may relate to the useof viral vectors, with regards to which mention is made of U.S. patentapplication Ser. No. 13/092,085, the contents of which are incorporatedby reference herein in their entirety. Tissue-specific regulatoryelements are known in the art and in this regard, mention is made ofU.S. Pat. No. 7,776,321, the contents of which are incorporated byreference herein in their entirety.

In some embodiments, a regulatory element is operably linked to one ormore elements of a CRISPR system so as to drive expression of the one ormore elements of the CRISPR system. In general, CRISPRs (ClusteredRegularly Interspaced Short Palindromic Repeats), also known as SPIDRs(SPacer Interspersed Direct Repeats), constitute a family of DNA locithat are usually specific to a particular bacterial species. The CRISPRlocus comprises a distinct class of interspersed short sequence repeats(SSRs) that were recognized in E. coli (Ishino et al., J. Bacteriol.,169:5429-5433 [1987]; and Nakata et al., J. Bacteriol., 171:3553-3556[1989]), and associated genes. Similar interspersed SSRs have beenidentified in Haloferax mediterranei, Streptococcus pyogenes, Anabaena,and Mycobacterium tuberculosis (See, Groenen et al., Mol. Microbiol.,10:1057-1065 [1993]; Hoe et al., Emerg. Infect. Dis., 5:254-263 [1999];Masepohl et al., Biochim. Biophys. Acta 1307:26-30 [1996]; and Mojica etal., Mol. Microbiol., 17:85-93 [1995]). The CRISPR loci typically differfrom other SSRs by the structure of the repeats, which have been termedshort regularly spaced repeats (SRSRs) (Janssen et al., OMICS J. Integ.Biol., 6:23-33 [2002]; and Mojica et al., Mol. Microbiol., 36:244-246[2000]). In general, the repeats are short elements that occur inclusters that are regularly spaced by unique intervening sequences witha substantially constant length (Mojica et al., [2000], supra). Althoughthe repeat sequences are highly conserved between strains, the number ofinterspersed repeats and the sequences of the spacer regions typicallydiffer from strain to strain (van Embden et al., J. Bacteriol.,182:2393-2401 [2000]). CRISPR loci have been identified in more than 40prokaryotes (See e.g., Jansen et al., Mol. Microbiol., 43:1565-1575[2002]; and Mojica et al., [2005]) including, but not limited toAeropyrum, Pyrobaculum, Sulfolobus, Archaeoglobus, Halocarcula,Methanobacterium, Methanococcus, Methanosarcina, Methanopyrus,Pyrococcus, Picrophilus, Thermoplasma, Corynebacterium, Mycobacterium,Streptomyces, Aquifex, Porphyromonas, Chlorobium, Thermus, Bacillus,Listeria, Staphylococcus, Clostridium, Thermoanaerobacter, Mycoplasma,Fusobacterium, Azarcus, Chromobacterium, Neisseria, Nitrosomonas,Desulfovibrio, Geobacter, Myxococcus, Campylobacter, Wolinella,Acinetobacter, Erwinia, Escherichia, Legionella, Methylococcus,Pasteurella, Photobacterium, Salmonella, Xanthomonas, Yersinia,Treponema, and Thermotoga.

In general, “CRISPR system” refers collectively to transcripts and otherelements involved in the expression of or directing the activity ofCRISPR-associated (“Cas”) genes, including sequences encoding a Casgene, a tracr (trans-activating CRISPR) sequence (e.g. tracrRNA or anactive partial tracrRNA), a tracr-mate sequence (encompassing a “directrepeat” and a tracrRNA-processed partial direct repeat in the context ofan endogenous CRISPR system), a guide sequence (also referred to as a“spacer” in the context of an endogenous CRISPR system), or othersequences and transcripts from a CRISPR locus. In embodiments of theinvention the terms guide sequence and guide RNA are usedinterchangeably. In some embodiments, one or more elements of a CRISPRsystem is derived from a type I, type II, or type III CRISPR system. Insome embodiments, one or more elements of a CRISPR system is derivedfrom a particular organism comprising an endogenous CRISPR system, suchas Streptococcus pyogenes. In general, a CRISPR system is characterizedby elements that promote the formation of a CRISPR complex at the siteof a target sequence (also referred to as a protospacer in the contextof an endogenous CRISPR system). In the context of formation of a CRISPRcomplex, “target sequence” refers to a sequence to which a guidesequence is designed to have complementarity, where hybridizationbetween a target sequence and a guide sequence promotes the formation ofa CRISPR complex. A target sequence may comprise any polynucleotide,such as DNA or RNA polynucleotides. In some embodiments, a targetsequence is located in the nucleus or cytoplasm of a cell.

In some embodiments, direct repeats may be identified in silico bysearching for repetitive motifs that fulfill any or all of the followingcriteria:

1. found in a 2 Kb window of genomic sequence flanking the type IICRISPR locus;

2. span from 20 to 50 bp; and

3. interspaced by 20 to 50 bp.

In some embodiments, 2 of these criteria may be used, for instance 1 and2, 2 and 3, or 1 and 3. In some embodiments, all 3 criteria may be used.

In some embodiments, candidate tracrRNA may be subsequently predicted bysequences that fulfill any or all of the following criteria:

1. sequence homology to direct repeats (motif search in Geneious with upto 18-bp mismatches);

2. presence of a predicted Rho-independent transcriptional terminator indirection of transcription; and

3. stable hairpin secondary structure between tracrRNA and directrepeat.

In some embodiments, 2 of these criteria may be used, for instance 1 and2, 2 and 3, or 1 and 3. In some embodiments, all 3 criteria may be used.

In some embodiments, chimeric synthetic guide RNAs (sgRNAs) designs mayincorporate at least 12 bp of duplex structure between the direct repeatand tracrRNA.

In preferred embodiments of the invention, the CRISPR system is a typeII CRISPR system and the Cas enzyme is Cas9, which catalyzes DNAcleavage. Enzymatic action by Cas9 derived from Streptococcus pyogenesor any closely related Cas9 generates double stranded breaks at targetsite sequences which hybridize to 20 nucleotides of the guide sequenceand that have a protospacer-adjacent motif (PAM) sequence (examplesinclude NGG/NRG or a PAM that can be determined as described herein)following the 20 nucleotides of the target sequence. CRISPR activitythrough Cas9 for site-specific DNA recognition and cleavage is definedby the guide sequence, the tracr sequence that hybridizes in part to theguide sequence and the PAM sequence. More aspects of the CRISPR systemare described in Karginov and Hannon, The CRISPR system: smallRNA-guided defence in bacteria and archaea, Mole Cell 2010, January 15;37(1): 7.

The type II CRISPR locus from Streptococcus pyogenes SF370, whichcontains a cluster of four genes Cas9, Cas1, Cas2, and Csn1, as well astwo non-coding RNA elements, tracrRNA and a characteristic array ofrepetitive sequences (direct repeats) interspaced by short stretches ofnon-repetitive sequences (spacers, about 30 bp each). In this system,targeted DNA double-strand break (DSB) is generated in four sequentialsteps (FIG. 2A). First, two non-coding RNAs, the pre-crRNA array andtracrRNA, are transcribed from the CRISPR locus. Second, tracrRNAhybridizes to the direct repeats of pre-crRNA, which is then processedinto mature crRNAs containing individual spacer sequences. Third, themature crRNA:tracrRNA complex directs Cas9 to the DNA target consistingof the protospacer and the corresponding PAM via heteroduplex formationbetween the spacer region of the crRNA and the protospacer DNA. Finally,Cas9 mediates cleavage of target DNA upstream of PAM to create a DSBwithin the protospacer (FIG. 2A). FIG. 2B demonstrates the nuclearlocalization of the codon optimized Cas9. To promote precisetranscriptional initiation, the RNA polymerase III-based U6 promoter wasselected to drive the expression of tracrRNA (FIG. 2C). Similarly, a U6promoter-based construct was developed to express a pre-crRNA arrayconsisting of a single spacer flanked by two direct repeats (DRs, alsoencompassed by the term “tracr-mate sequences”; FIG. 2C). The initialspacer was designed to target a 33-base-pair (bp) target site (30-bpprotospacer plus a 3-bp CRISPR motif (PAM) sequence satisfying the NGGrecognition motif of Cas9) in the human EMX1 locus (FIG. 2C), a key genein the development of the cerebral cortex.

Typically, in the context of an endogenous CRISPR system, formation of aCRISPR complex (comprising a guide sequence hybridized to a targetsequence and complexed with one or more Cas proteins) results incleavage of one or both strands in or near (e.g. within 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence.Without wishing to be bound by theory, the tracr sequence, which maycomprise or consist of all or a portion of a wild-type tracr sequence(e.g. about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85, ormore nucleotides of a wild-type tracr sequence), may also form part of aCRISPR complex, such as by hybridization along at least a portion of thetracr sequence to all or a portion of a tracr mate sequence that isoperably linked to the guide sequence. In some embodiments, one or morevectors driving expression of one or more elements of a CRISPR systemare introduced into a host cell such that expression of the elements ofthe CRISPR system direct formation of a CRISPR complex at one or moretarget sites. For example, a Cas enzyme, a guide sequence linked to atracr-mate sequence, and a tracr sequence could each be operably linkedto separate regulatory elements on separate vectors. Alternatively, twoor more of the elements expressed from the same or different regulatoryelements, may be combined in a single vector, with one or moreadditional vectors providing any components of the CRISPR system notincluded in the first vector. CRISPR system elements that are combinedin a single vector may be arranged in any suitable orientation, such asone element located 5′ with respect to (“upstream” of) or 3′ withrespect to (“downstream” of) a second element. The coding sequence ofone element may be located on the same or opposite strand of the codingsequence of a second element, and oriented in the same or oppositedirection. In some embodiments, a single promoter drives expression of atranscript encoding a CRISPR enzyme and one or more of the guidesequence, tracr mate sequence (optionally operably linked to the guidesequence), and a tracr sequence embedded within one or more intronsequences (e.g. each in a different intron, two or more in at least oneintron, or all in a single intron). In some embodiments, the CRISPRenzyme, guide sequence, tracr mate sequence, and tracr sequence areoperably linked to and expressed from the same promoter.

In some embodiments, a vector comprises one or more insertion sites,such as a restriction endonuclease recognition sequence (also referredto as a “cloning site”). In some embodiments, one or more insertionsites (e.g. about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore insertion sites) are located upstream and/or downstream of one ormore sequence elements of one or more vectors. In some embodiments, avector comprises an insertion site upstream of a tracr mate sequence,and optionally downstream of a regulatory element operably linked to thetracr mate sequence, such that following insertion of a guide sequenceinto the insertion site and upon expression the guide sequence directssequence-specific binding of a CRISPR complex to a target sequence in aeukaryotic cell. In some embodiments, a vector comprises two or moreinsertion sites, each insertion site being located between two tracrmate sequences so as to allow insertion of a guide sequence at eachsite. In such an arrangement, the two or more guide sequences maycomprise two or more copies of a single guide sequence, two or moredifferent guide sequences, or combinations of these. When multipledifferent guide sequences are used, a single expression construct may beused to target CRISPR activity to multiple different, correspondingtarget sequences within a cell. For example, a single vector maycomprise about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,or more guide sequences. In some embodiments, about or more than about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more such guide-sequence-containingvectors may be provided, and optionally delivered to a cell.

In some embodiments, a vector comprises a regulatory element operablylinked to an enzyme-coding sequence encoding a CRISPR enzyme, such as aCas protein. Non-limiting examples of Cas proteins include Cas1, Cas1B,Cas2, Cas3, Cas4, Cas5, Cash, Cas7, Cas8, Cas9 (also known as Csn1 andCsx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2,Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2,Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2,Csf3, Csf4, homologues thereof, or modified versions thereof. In someembodiments, the unmodified CRISPR enzyme has DNA cleavage activity,such as Cas9. In some embodiments, the CRISPR enzyme directs cleavage ofone or both strands at the location of a target sequence, such as withinthe target sequence and/or within the complement of the target sequence.In some embodiments, the CRISPR enzyme directs cleavage of one or bothstrands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100,200, 500, or more base pairs from the first or last nucleotide of atarget sequence. In some embodiments, a vector encodes a CRISPR enzymethat is mutated to with respect to a corresponding wild-type enzyme suchthat the mutated CRISPR enzyme lacks the ability to cleave one or bothstrands of a target polynucleotide containing a target sequence. Forexample, an aspartate-to-alanine substitution (D10A) in the RuvC Icatalytic domain of Cas9 from S. pyogenes converts Cas9 from a nucleasethat cleaves both strands to a nickase (cleaves a single strand). Otherexamples of mutations that render Cas9 a nickase include, withoutlimitation, H840A, N854A, and N863A. As a further example, two or morecatalytic domains of Cas9 (RuvC I, RuvC II, and RuvC III or the HNHdomain) may be mutated to produce a mutated Cas9 substantially lackingall DNA cleavage activity. In some embodiments, a D10A mutation iscombined with one or more of H840A, N854A, or N863A mutations to producea Cas9 enzyme substantially lacking all DNA cleavage activity. In someembodiments, a CRISPR enzyme is considered to substantially lack all DNAcleavage activity when the DNA cleavage activity of the mutated enzymeis less than about 25%, 10%, 5%, 1%, 0.1%, 0.01%, or lower with respectto its non-mutated form.

An aspartate-to-alanine substitution (D10A) in the RuvC I catalyticdomain of SpCas9 was engineered to convert the nuclease into a nickase(SpCas9n) (see e.g. Sapranauskas et al., 2011, Nucleic Acis Research,39: 9275; Gasiunas et al., 2012, Proc. Natl. Acad. Sci. USA, 109:E2579),such that nicked genomic DNA undergoes the high-fidelityhomology-directed repair (HDR). Surveyor assay confirmed that SpCas9ndoes not generate indels at the EMX1 protospacer target. Co-expressionof EMX1-targeting chimeric crRNA (having the tracrRNA component as well)with SpCas9 produced indels in the target site, whereas co-expressionwith SpCas9n did not (n=3). Moreover, sequencing of 327 amplicons didnot detect any indels induced by SpCas9n. The same locus was selected totest CRISPR-mediated HR by co-transfecting HEK 293FT cells with thechimeric RNA targeting EMX1, hSpCas9 or hSpCas9n, as well as a HRtemplate to introduce a pair of restriction sites (HindIll and NheI)near the protospacer.

Preferred orthologs are described herein. A Cas enzyme may be identifiedCas9 as this can refer to the general class of enzymes that sharehomology to the biggest nuclease with multiple nuclease domains from thetype II CRISPR system. Most preferably, the Cas9 enzyme is from, or isderived from, spCas9 or saCas9. By derived, Applicants mean that thederived enzyme is largely based, in the sense of having a high degree ofsequence homology with, a wildtype enzyme, but that it has been mutated(modified) in some way as described herein.

It will be appreciated that the terms Cas and CRISPR enzyme aregenerally used herein interchangeably, unless otherwise apparent. Asmentioned above, many of the residue numberings used herein refer to theCas9 enzyme from the type II CRISPR locus in Streptococcus pyogenes.However, it will be appreciated that this invention includes many moreCas9s from other species of microbes, such as SpCas9, SaCa9, StlCas9 andso forth.

An example of a codon optimized sequence, in this instance optimized forhumans (i.e. being optimized for expression in humans) is providedherein, see the SaCas9 human codon optimized sequence. Whilst this ispreferred, it will be appreciated that other examples are possible andcodon optimization for a host species other than human, or for codonoptimization for specific organs such as the brain, is known.

In some embodiments, an enzyme coding sequence encoding a CRISPR enzymeis codon optimized for expression in particular cells, such aseukaryotic cells. The eukaryotic cells may be those of or derived from aparticular organism, such as a mammal, including but not limited tohuman, mouse, rat, rabbit, dog, or non-human mammal or primate. In someembodiments, processes for modifying the germ line genetic identity ofhuman beings and/or processes for modifying the genetic identity ofanimals which are likely to cause them suffering without any substantialmedical benefit to man or animal, and also animals resulting from suchprocesses, may be excluded.

In general, codon optimization refers to a process of modifying anucleic acid sequence for enhanced expression in the host cells ofinterest by replacing at least one codon (e.g. about or more than about1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the nativesequence with codons that are more frequently or most frequently used inthe genes of that host cell while maintaining the native amino acidsequence. Various species exhibit particular bias for certain codons ofa particular amino acid. Codon bias (differences in codon usage betweenorganisms) often correlates with the efficiency of translation ofmessenger RNA (mRNA), which is in turn believed to be dependent on,among other things, the properties of the codons being translated andthe availability of particular transfer RNA (tRNA) molecules. Thepredominance of selected tRNAs in a cell is generally a reflection ofthe codons used most frequently in peptide synthesis. Accordingly, genescan be tailored for optimal gene expression in a given organism based oncodon optimization. Codon usage tables are readily available, forexample, at the “Codon Usage Database” available atwww.kazusa.orjp/codon/ (visited Jul. 9, 2002), and these tables can beadapted in a number of ways. See Nakamura, Y., et al. “Codon usagetabulated from the international DNA sequence databases: status for theyear 2000” Nucl. Acids Res. 28:292 (2000). Computer algorithms for codonoptimizing a particular sequence for expression in a particular hostcell are also available, such as Gene Forge (Aptagen; Jacobus, P A), arealso available. In some embodiments, one or more codons (e.g. 1, 2, 3,4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence encodinga CRISPR enzyme correspond to the most frequently used codon for aparticular amino acid.

In some embodiments, a vector encodes a CRISPR enzyme comprising one ormore nuclear localization sequences (NLSs), such as about or more thanabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs. In some embodiments,the CRISPR enzyme comprises about or more than about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or more NLSs at or near the amino-terminus, about or morethan about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near thecarboxy-terminus, or a combination of these (e.g. one or more NLS at theamino-terminus and one or more NLS at the carboxy terminus). When morethan one NLS is present, each may be selected independently of theothers, such that a single NLS may be present in more than one copyand/or in combination with one or more other NLSs present in one or morecopies. In a preferred embodiment of the invention, the CRISPR enzymecomprises at most 6 NLSs. In some embodiments, an NLS is considered nearthe N- or C-terminus when the nearest amino acid of the NLS is withinabout 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more amino acidsalong the polypeptide chain from the N- or C-terminus. Non-limitingexamples of NLSs include an NLS sequence derived from: the NLS of theSV40 virus large T-antigen, having the amino acid sequence PKKKRKV (SEQID NO: 60); the NLS from nucleoplasmin (e.g. the nucleoplasmin bipartiteNLS with the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 61)); the c-myc NLShaving the amino acid sequence PAAKRVKLD (SEQ ID NO: 62) or RQRRNELKRSP(SEQ ID NO: 63); the hRNPA1 M9 NLS having the sequenceNQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 64); the sequenceRMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 65) of the IBBdomain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO: 66) andPPKKARED (SEQ ID NO: 67) of the myoma T protein; the sequence PQPKKKPL(SEQ ID NO: 68) of human p53; the sequence SALIKKKKKMAP (SEQ ID NO: 69)of mouse c-abl IV; the sequences DRLRR (SEQ ID NO: 70) and PKQKKRK (SEQID NO: 71) of the influenza virus NS1; the sequence RKLKKKIKKL (SEQ IDNO: 72) of the Hepatitis virus delta antigen; the sequence REKKKFLKRR(SEQ ID NO: 73) of the mouse Mxl protein; the sequenceKRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 74) of the human poly(ADP-ribose)polymerase; and the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 75) of thesteroid hormone receptors (human) glucocorticoid.

In general, the one or more NLSs are of sufficient strength to driveaccumulation of the CRISPR enzyme in a detectable amount in the nucleusof a eukaryotic cell. In general, strength of nuclear localizationactivity may derive from the number of NLSs in the CRISPR enzyme, theparticular NLS(s) used, or a combination of these factors. Detection ofaccumulation in the nucleus may be performed by any suitable technique.For example, a detectable marker may be fused to the CRISPR enzyme, suchthat location within a cell may be visualized, such as in combinationwith a means for detecting the location of the nucleus (e.g. a stainspecific for the nucleus such as DAPI). Cell nuclei may also be isolatedfrom cells, the contents of which may then be analyzed by any suitableprocess for detecting protein, such as immunohistochemistry, Westernblot, or enzyme activity assay. Accumulation in the nucleus may also bedetermined indirectly, such as by an assay for the effect of CRISPRcomplex formation (e.g. assay for DNA cleavage or mutation at the targetsequence, or assay for altered gene expression activity affected byCRISPR complex formation and/or CRISPR enzyme activity), as compared toa control no exposed to the CRISPR enzyme or complex, or exposed to aCRISPR enzyme lacking the one or more NLSs.

In general, a guide sequence is any polynucleotide sequence havingsufficient complementarity with a target polynucleotide sequence tohybridize with the target sequence and direct sequence-specific bindingof a CRISPR complex to the target sequence. In some embodiments, thedegree of complementarity between a guide sequence and its correspondingtarget sequence, when optimally aligned using a suitable alignmentalgorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%,95%, 97.5%, 99%, or more. Optimal alignment may be determined with theuse of any suitable algorithm for aligning sequences, non-limitingexample of which include the Smith-Waterman algorithm, theNeedleman-Wunsch algorithm, algorithms based on the Burrows-WheelerTransform (e.g. the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT,Novoalign (Novocraft Technologies; available at www.novocraft.com),ELAND (Illumina, San Diego, Calif.), SOAP (available atsoap.genomics.org.cn), and Maq (available at maq.sourceforge.net). Insome embodiments, a guide sequence is about or more than about 5, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In someembodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30,25, 20, 15, 12, or fewer nucleotides in length. The ability of a guidesequence to direct sequence-specific binding of a CRISPR complex to atarget sequence may be assessed by any suitable assay. For example, thecomponents of a CRISPR system sufficient to form a CRISPR complex,including the guide sequence to be tested, may be provided to a hostcell having the corresponding target sequence, such as by transfectionwith vectors encoding the components of the CRISPR sequence, followed byan assessment of preferential cleavage within the target sequence, suchas by Surveyor assay as described herein. Similarly, cleavage of atarget polynucleotide sequence may be evaluated in a test tube byproviding the target sequence, components of a CRISPR complex, includingthe guide sequence to be tested and a control guide sequence differentfrom the test guide sequence, and comparing binding or rate of cleavageat the target sequence between the test and control guide sequencereactions. Other assays are possible, and will occur to those skilled inthe art.

A guide sequence may be selected to target any target sequence. In someembodiments, the target sequence is a sequence within a genome of acell. Exemplary target sequences include those that are unique in thetarget genome. For example, for the S. pyogenes Cas9, a unique targetsequence in a genome may include a Cas9 target site of the formMMMMMMMMNNNNNNNNNNNNXGG where NNNNNNNNNNNNXGG (N is A, G, T, or C; and Xcan be anything) has a single occurrence in the genome. A unique targetsequence in a genome may include an S. pyogenes Cas9 target site of theform MMMMMMMMMNNNNNNNNNNXGG where NNNNNNNNNNNXGG (N is A, G, T, or C;and X can be anything) has a single occurrence in the genome. For the S.thermophilus CRISPR1 Cas9, a unique target sequence in a genome mayinclude a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXXAGAAW (SEQID NO: 76) where NNNNNNNNNNNNXXAGAAW (SEQ ID NO: 77) (N is A, G, T, orC; X can be anything; and W is A or T) has a single occurrence in thegenome. A unique target sequence in a genome may include an S.thermophilus CRISPR1 Cas9 target site of the formMMMMMMMMMNNNNNNNNNNNXXAGAAW (SEQ ID NO: 78) where NNNNNNNNNNNXXAGAAW(SEQ ID NO: 79) (N is A, G, T, or C; X can be anything; and W is A or T)has a single occurrence in the genome. For the S. pyogenes Cas9, aunique target sequence in a genome may include a Cas9 target site of theform MMMMMMMMNNNNNNNNNNNXGGXG where NNNNNNNNNNNNXGGXG (N is A, G, T, orC; and X can be anything) has a single occurrence in the genome. Aunique target sequence in a genome may include an S. pyogenes Cas9target site of the form MMMMMMMMMNNNNNNNNNNNXGGXG where NNNNNNNNNNNXGGXG(N is A, G, T, or C; and X can be anything) has a single occurrence inthe genome. In each of these sequences “M” may be A, G, T, or C, andneed not be considered in identifying a sequence as unique.

In some embodiments, a guide sequence is selected to reduce the degreesecondary structure within the guide sequence. In some embodiments,about or less than about 75%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1%,or fewer of the nucleotides of the guide sequence participate inself-complementary base pairing when optimally folded. Optimal foldingmay be determined by any suitable polynucleotide folding algorithm. Someprograms are based on calculating the minimal Gibbs free energy. Anexample of one such algorithm is mFold, as described by Zuker andStiegler (Nucleic Acids Res. 9 (1981), 133-148). Another example foldingalgorithm is the online webserver RNAfold, developed at Institute forTheoretical Chemistry at the University of Vienna, using the centroidstructure prediction algorithm (see e.g. A. R. Gruber et al., 2008, Cell106(1): 23-24; and PA Carr and GM Church, 2009, Nature Biotechnology27(12): 1151-62).

In general, a tracr mate sequence includes any sequence that hassufficient complementarity with a tracr sequence to promote one or moreof: (1) excision of a guide sequence flanked by tracr mate sequences ina cell containing the corresponding tracr sequence; and (2) formation ofa CRISPR complex at a target sequence, wherein the CRISPR complexcomprises the tracr mate sequence hybridized to the tracr sequence. Ingeneral, degree of complementarity is with reference to the optimalalignment of the tracr mate sequence and tracr sequence, along thelength of the shorter of the two sequences. Optimal alignment may bedetermined by any suitable alignment algorithm, and may further accountfor secondary structures, such as self-complementarity within either thetracr sequence or tracr mate sequence. In some embodiments, the degreeof complementarity between the tracr sequence and tracr mate sequencealong the length of the shorter of the two when optimally aligned isabout or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,97.5%, 99%, or higher. In some embodiments, the tracr sequence is aboutor more than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 25, 30, 40, 50, or more nucleotides in length. In someembodiments, the tracr sequence and tracr mate sequence are containedwithin a single transcript, such that hybridization between the twoproduces a transcript having a secondary structure, such as a hairpin.In an embodiment of the invention, the transcript or transcribedpolynucleotide sequence has at least two or more hairpins. In preferredembodiments, the transcript has two, three, four or five hairpins. In afurther embodiment of the invention, the transcript has at most fivehairpins. In a hairpin structure the portion of the sequence 5′ of thefinal “N” and upstream of the loop corresponds to the tracr matesequence, and the portion of the sequence 3′ of the loop corresponds tothe tracr sequence Further non-limiting examples of singlepolynucleotides comprising a guide sequence, a tracr mate sequence, anda tracr sequence are as follows (listed 5′ to 3′), where “N” representsa base of a guide sequence, the first block of lower case lettersrepresent the tracr mate sequence, and the second block of lower caseletters represent the tracr sequence, and the final poly-T sequencerepresents the transcription terminator: (1)NNNNNNNNNNNNNNNNNNNNNgtttttgtactctcaagatttaGAAAtaaatcttgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT (SEQ IDNO: 80); (2)NNNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT (SEQ ID NO: 81);(3)NNNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgtTTTTTT (SEQ ID NO: 82); (4)NNNNNNNNNNNNNNNNNNNNNgttttagagctaGAAAtagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcTTTTTT (SEQ ID NO: 83); (5)NNNNNNNNNNNNNNNNNNNNNgttttagagctaGAAATAGcaagttaaaataaggctagtccgttatcaacttgaaaaagtgTTTTTTT (SEQ ID NO: 84); and (6)gttttagagctagAAATAGcaagttaaaataaggctagtccgttatcaTT TTTTTT (SEQ ID NO:85). In some embodiments, sequences (1) to (3) are used in combinationwith Cas9 from S. thermophilus CRISPR1. In some embodiments, sequences(4) to (6) are used in combination with Cas9 from S. pyogenes. In someembodiments, the tracr sequence is a separate transcript from atranscript comprising the tracr mate sequence.

In some embodiments, a recombination template is also provided. Arecombination template may be a component of another vector as describedherein, contained in a separate vector, or provided as a separatepolynucleotide. In some embodiments, a recombination template isdesigned to serve as a template in homologous recombination, such aswithin or near a target sequence nicked or cleaved by a CRISPR enzyme asa part of a CRISPR complex. A template polynucleotide may be of anysuitable length, such as about or more than about 10, 15, 20, 25, 50,75, 100, 150, 200, 500, 1000, or more nucleotides in length. In someembodiments, the template polynucleotide is complementary to a portionof a polynucleotide comprising the target sequence. When optimallyaligned, a template polynucleotide might overlap with one or morenucleotides of a target sequences (e.g. about or more than about 1, 5,10, 15, 20, or more nucleotides). In some embodiments, when a templatesequence and a polynucleotide comprising a target sequence are optimallyaligned, the nearest nucleotide of the template polynucleotide is withinabout 1, 5, 10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500, 1000, 5000,10000, or more nucleotides from the target sequence.

In some embodiments, the CRISPR enzyme is part of a fusion proteincomprising one or more heterologous protein domains (e.g. about or morethan about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains in addition tothe CRISPR enzyme). A CRISPR enzyme fusion protein may comprise anyadditional protein sequence, and optionally a linker sequence betweenany two domains. Examples of protein domains that may be fused to aCRISPR enzyme include, without limitation, epitope tags, reporter genesequences, and protein domains having one or more of the followingactivities: methylase activity, demethylase activity, transcriptionactivation activity, transcription repression activity, transcriptionrelease factor activity, histone modification activity, RNA cleavageactivity and nucleic acid binding activity. Non-limiting examples ofepitope tags include histidine (His) tags, V5 tags, FLAG tags, influenzahemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx)tags. Examples of reporter genes include, but are not limited to,glutathione-S-transferase (GST), horseradish peroxidase (HRP),chloramphenicol acetyltransferase (CAT) beta-galactosidase,beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed,DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP),and autofluorescent proteins including blue fluorescent protein (BFP). ACRISPR enzyme may be fused to a gene sequence encoding a protein or afragment of a protein that bind DNA molecules or bind other cellularmolecules, including but not limited to maltose binding protein (MBP),S-tag, Lex A DNA binding domain (DBD) fusions, GAL4 DNA binding domainfusions, and herpes simplex virus (HSV) BP16 protein fusions. Additionaldomains that may form part of a fusion protein comprising a CRISPRenzyme are described in US20110059502, incorporated herein by reference.In some embodiments, a tagged CRISPR enzyme is used to identify thelocation of a target sequence.

In some embodiments, a CRISPR enzyme may form a component of aninducible system. The inducible nature of the system would allow forspatiotemporal control of gene editing or gene expression using a formof energy. The form of energy may include but is not limited toelectromagnetic radiation, sound energy, chemical energy and thermalenergy. Examples of inducible system include tetracycline induciblepromoters (Tet-On or Tet-Off), small molecule two-hybrid transcriptionactivations systems (FKBP, ABA, etc), or light inducible systems(Phytochrome, LOV domains, or cryptochrome). In one embodiment, theCRISPR enzyme may be a part of a Light Inducible TranscriptionalEffector (LITE) to direct changes in transcriptional activity in asequence-specific manner. The components of a light may include a CRISPRenzyme, a light-responsive cytochrome heterodimer (e.g. from Arabidopsisthaliana), and a transcriptional activation/repression domain. Furtherexamples of inducible DNA binding proteins and methods for their use areprovided in U.S. 61/736,465 and U.S. 61/721,283, which is herebyincorporated by reference in its entirety.

In some aspects, the invention provides methods comprising deliveringone or more polynucleotides, such as or one or more vectors as describedherein, one or more transcripts thereof, and/or one or proteinstranscribed therefrom, to a host cell. In some aspects, the inventionfurther provides cells produced by such methods, and animals comprisingor produced from such cells. In some embodiments, a CRISPR enzyme incombination with (and optionally complexed with) a guide sequence isdelivered to a cell. Conventional viral and non-viral based genetransfer methods can be used to introduce nucleic acids in mammaliancells or target tissues. Such methods can be used to administer nucleicacids encoding components of a CRISPR system to cells in culture, or ina host organism. Non-viral vector delivery systems include DNA plasmids,RNA (e.g. a transcript of a vector described herein), naked nucleicacid, and nucleic acid complexed with a delivery vehicle, such as aliposome. Viral vector delivery systems include DNA and RNA viruses,which have either episomal or integrated genomes after delivery to thecell. For a review of gene therapy procedures, see Anderson, Science256:808-813 (1992); Nabel & Felgner, TIBTECH 11:211-217 (1993); Mitani &Caskey, TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993);Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology6(10):1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience8:35-36 (1995); Kremer & Perricaudet, British Medical Bulletin51(1):31-44 (1995); Haddada et al., in Current Topics in Microbiologyand Immunology Doerfler and Bohm (eds) (1995); and Yu et al., GeneTherapy 1:13-26 (1994).

Methods of non-viral delivery of nucleic acids include lipofection,microinjection, biolistics, virosomes, liposomes, immunoliposomes,polycation or lipid:nucleic acid conjugates, naked DNA, artificialvirions, and agent-enhanced uptake of DNA. Lipofection is described ine.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) andlipofection reagents are sold commercially (e.g., Transfectam™ andLipofectin™). Cationic and neutral lipids that are suitable forefficient receptor-recognition lipofection of polynucleotides includethose of Felgner, WO 91/17424; WO 91/16024. Delivery can be to cells(e.g. in vitro or ex vivo administration) or target tissues (e.g. invivo administration).

The preparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese etal., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem.5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gaoet al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res.52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871,4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

The use of RNA or DNA viral based systems for the delivery of nucleicacids take advantage of highly evolved processes for targeting a virusto specific cells in the body and trafficking the viral payload to thenucleus. Viral vectors can be administered directly to patients (invivo) or they can be used to treat cells in vitro, and the modifiedcells may optionally be administered to patients (ex vivo). Conventionalviral based systems could include retroviral, lentivirus, adenoviral,adeno-associated and herpes simplex virus vectors for gene transfer.Integration in the host genome is possible with the retrovirus,lentivirus, and adeno-associated virus gene transfer methods, oftenresulting in long term expression of the inserted transgene.Additionally, high transduction efficiencies have been observed in manydifferent cell types and target tissues.

The tropism of a retrovirus can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vectors that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system would thereforedepend on the target tissue. Retroviral vectors are comprised ofcis-acting long terminal repeats with packaging capacity for up to 6-10kb of foreign sequence. The minimum cis-acting LTRs are sufficient forreplication and packaging of the vectors, which are then used tointegrate the therapeutic gene into the target cell to provide permanenttransgene expression. Widely used retroviral vectors include those basedupon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV),Simian Immuno deficiency virus (SIV), human immuno deficiency virus(HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol.66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992);Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol.63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991);PCT/US94/05700).

In applications where transient expression is preferred, adenoviralbased systems may be used. Adenoviral based vectors are capable of veryhigh transduction efficiency in many cell types and do not require celldivision. With such vectors, high titer and levels of expression havebeen obtained. This vector can be produced in large quantities in arelatively simple system.

Adeno-associated virus (“AAV”) vectors may also be used to transducecells with target nucleic acids, e.g., in the in vitro production ofnucleic acids and peptides, and for in vivo and ex vivo gene therapyprocedures (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat.No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994);Muzyczka, J. Clin. Invest. 94:1351 (1994). Construction of recombinantAAV vectors are described in a number of publications, including U.S.Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260(1985); Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat& Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., J. Virol.63:03822-3828 (1989).

Packaging cells are typically used to form virus particles that arecapable of infecting a host cell. Such cells include 293 cells, whichpackage adenovirus, and w2 cells or PA317 cells, which packageretrovirus. Viral vectors used in gene therapy are usually generated byproducer a cell line that packages a nucleic acid vector into a viralparticle. The vectors typically contain the minimal viral sequencesrequired for packaging and subsequent integration into a host, otherviral sequences being replaced by an expression cassette for thepolynucleotide(s) to be expressed. The missing viral functions aretypically supplied in trans by the packaging cell line. For example, AAVvectors used in gene therapy typically only possess ITR sequences fromthe AAV genome which are required for packaging and integration into thehost genome. Viral DNA is packaged in a cell line, which contains ahelper plasmid encoding the other AAV genes, namely rep and cap, butlacking ITR sequences. The cell line may also infected with adenovirusas a helper. The helper virus promotes replication of the AAV vector andexpression of AAV genes from the helper plasmid. The helper plasmid isnot packaged in significant amounts due to a lack of ITR sequences.Contamination with adenovirus can be reduced by, e.g., heat treatment towhich adenovirus is more sensitive than AAV.

Accordingly, AAV is considered an ideal candidate for use as atransducing vector. Such AAV transducing vectors can comprise sufficientcis-acting functions to replicate in the presence of adenovirus orherpesvirus or poxvirus (e.g., vaccinia virus) helper functions providedin trans. Recombinant AAV (rAAV) can be used to carry exogenous genesinto cells of a variety of lineages. In these vectors, the AAV capand/or rep genes are deleted from the viral genome and replaced with aDNA segment of choice. Current AAV vectors may accommodate up to 4300bases of inserted DNA.

There are a number of ways to produce rAAV, and the invention providesrAAV and methods for preparing rAAV. For example, plasmid(s) containingor consisting essentially of the desired viral construct are transfectedinto AAV-infected cells. In addition, a second or additional helperplasmid is cotransfected into these cells to provide the AAV rep and/orcap genes which are obligatory for replication and packaging of therecombinant viral construct. Under these conditions, the rep and/or capproteins of AAV act in trans to stimulate replication and packaging ofthe rAAV construct. Two to Three days after transfection, rAAV isharvested. Traditionally rAAV is harvested from the cells along withadenovirus. The contaminating adenovirus is then inactivated by heattreatment. In the instant invention, rAAV is advantageously harvestednot from the cells themselves, but from cell supernatant. Accordingly,in an initial aspect the invention provides for preparing rAAV, and inaddition to the foregoing, rAAV can be prepared by a method thatcomprises or consists essentially of: infecting susceptible cells with arAAV containing exogenous DNA including DNA for expression, and helpervirus (e.g., adenovirus, herpesvirus, poxvirus such as vaccinia virus)wherein the rAAV lacks functioning cap and/or rep (and the helper virus(e.g., adenovirus, herpesvirus, poxvirus such as vaccinia virus)provides the cap and/or rev function that the rAAV lacks); or infectingsusceptible cells with a rAAV containing exogenous DNA including DNA forexpression, wherein the recombinant lacks functioning cap and/or rep,and transfecting said cells with a plasmid supplying cap and/or repfunction that the rAAV lacks; or infecting susceptible cells with a rAAVcontaining exogenous DNA including DNA for expression, wherein therecombinant lacks functioning cap and/or rep, wherein said cells supplycap and/or rep function that the recombinant lacks; or transfecting thesusceptible cells with an AAV lacking functioning cap and/or rep andplasmids for inserting exogenous DNA into the recombinant so that theexogenous DNA is expressed by the recombinant and for supplying repand/or cap functions whereby transfection results in an rAAV containingthe exogenous DNA including DNA for expression that lacks functioningcap and/or rep.

The rAAV can be from an AAV as herein described, and advantageously canbe an rAAV1, rAAV2, AAV5 or rAAV having hybrid or capsid which maycomprise AAV1, AAV2, AAV5 or any combination thereof. One can select theAAV of the rAAV with regard to the cells to be targeted by the rAAV;e.g., one can select AAV serotypes 1, 2, 5 or a hybrid or capsid AAV1,AAV2, AAV5 or any combination thereof for targeting brain or neuronalcells; and one can select AAV4 for targeting cardiac tissue.

In addition to 293 cells, other cells that can be used in the practiceof the invention and the relative infectivity of certain AAV serotypesin vitro as to these cells (see Grimm, D. et al, J. Virol. 82: 5887-5911(2008)) are as follows:

Cell Line AAV-1 AAV-2 AAV-3 AAV-4 AAV-5 AAV-6 AAV-8 AAV-9 Huh-7 13 1002.5 0.0 0.1 10 0.7 0.0 HEK293 25 100 2.5 0.1 0.1 5 0.7 0.1 HeLa 3 1002.0 0.1 6.7 1 0.2 0.1 HepG2 3 100 16.7 0.3 1.7 5 0.3 ND Hep1A 20 100 0.21.0 0.1 1 0.2 0.0 911 17 100 11 0.2 0.1 17 0.1 ND CHO 100 100 14 1.4 33350 10 1.0 COS 33 100 33 3.3 5.0 14 2.0 0.5 MeWo 10 100 20 0.3 6.7 10 1.00.2 NIH3T3 10 100 2.9 2.9 0.3 10 0.3 ND A549 14 100 20 ND 0.5 10 0.5 0.1HT1180 20 100 10 0.1 0.3 33 0.5 0.1 Monocytes 1111 100 ND ND 125 1429 NDND Immature DC 2500 100 ND ND 222 2857 ND ND Mature DC 2222 100 ND ND333 3333 ND ND

The invention provides rAAV that contains or consists essentially of anexogenous nucleic acid molecule encoding a CRISPR (Clustered RegularlyInterspaced Short Palindromic Repeats) system, e.g., a plurality ofcassettes comprising or consisting a first cassette comprising orconsisting essentially of a promoter, a nucleic acid molecule encoding aCRISPR-associated (Cas) protein (putative nuclease or helicaseproteins), e.g., Cas9 and a terminator, and a two, or more,advantageously up to the packaging size limit of the vector, e.g., intotal (including the first cassette) five, cassettes comprising orconsisting essentially of a promoter, nucleic acid molecule encodingguide RNA (gRNA) and a terminator (e.g., each cassette schematicallyrepresented as Promoter-gRNA1-terminator, Promoter-gRNA2-terminatorPromoter-gRNA(N)-terminator (where N is a number that can be insertedthat is at an upper limit of the packaging size limit of the vector), ortwo or more individual rAAVs, each containing one or more than onecassette of a CRISPR system, e.g., a first rAAV containing the firstcassette comprising or consisting essentially of a promoter, a nucleicacid molecule encoding Cas, e.g., Cas9 and a terminator, and a secondrAAV containing a plurality, four, cassettes comprising or consistingessentially of a promoter, nucleic acid molecule encoding guide RNA(gRNA) and a terminator (e.g., each cassette schematically representedas Promoter-gRNA1-terminator, Promoter-gRNA2-terminator . . .Promoter-gRNA(N)-terminator (where N is a number that can be insertedthat is at an upper limit of the packaging size limit of the vector). AsrAAV is a DNA virus, the nucleic acid molecules in the herein discussionconcerning AAV or rAAV are advantageously DNA. The promoter is in someembodiments advantageously human Synapsin I promoter (hSyn).

Additional methods for the delivery of nucleic acids to cells are knownto those skilled in the art. See, for example, US20030087817,incorporated herein by reference.

In some embodiments, a host cell is transiently or non-transientlytransfected with one or more vectors described herein. In someembodiments, a cell is transfected as it naturally occurs in a subject.In some embodiments, a cell that is transfected is taken from a subject.In some embodiments, the cell is derived from cells taken from asubject, such as a cell line. A wide variety of cell lines for tissueculture are known in the art. Examples of cell lines include, but arenot limited to, C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa-S3, Huh1,Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Pancl, PC-3, TF1,CTLL-2, C1R, Rath, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calul, SW480,SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB55,Jurkat, J45.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E,MRCS, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6, COS-M6A, BS-C-1monkey kidney epithelial, BALB/3T3 mouse embryo fibroblast, 3T3 Swiss,3T3-L1, 132-d5 human fetal fibroblasts; 10.1 mouse fibroblasts, 293-T,3T3, 721, 9 L, A2780, A2780ADR, A2780cis, A172, A20, A253, A431, A-549,ALC, B16, B35, BCP-1 cells, BEAS-2B, bEnd.3, BHK-21, BR 293, BxPC3,C3H-10T1/2, C6/36, Cal-27, CHO, CHO-7, CHO-IR, CHO-K1, CHO-K2, CHO-T,CHO Dhfr −/−, COR-L23, COR-L23/CPR, COR-L23/5010, COR-L23/R23, COS-7,COV-434, CML T1, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3,EMT6/AR1, EMT6/AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa,Hepalc1c7, HL-60, HMEC, HT-29, Jurkat, JY cells, K562 cells, Ku812,KCL22, KG1, KYO1, LNCap, Ma-Mel 1-48, MC-38, MCF-7, MCF-10A, MDA-MB-231,MDA-MB-468, MDA-MB-435, MDCK II, MDCK II, MOR/0.2R, MONO-MAC 6, MTD-1A,MyEnd, NCI-H69/CPR, NCI-H69/LX10, NCI-H69/LX20, NCI-H69/LX4, NIH-3T3,NALM-1, NW-145, OPCN/OPCT cell lines, Peer, PNT-1A/PNT 2, RenCa, RIN-5F,RMA/RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell line,U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-1, YAR, andtransgenic varieties thereof. Cell lines are available from a variety ofsources known to those with skill in the art (see, e.g., the AmericanType Culture Collection (ATCC) (Manassas, Va.)). In some embodiments, acell transfected with one or more vectors described herein is used toestablish a new cell line comprising one or more vector-derivedsequences. In some embodiments, a cell transiently transfected with thecomponents of a CRISPR system as described herein (such as by transienttransfection of one or more vectors, or transfection with RNA), andmodified through the activity of a CRISPR complex, is used to establisha new cell line comprising cells containing the modification but lackingany other exogenous sequence. In some embodiments, cells transiently ornon-transiently transfected with one or more vectors described herein,or cell lines derived from such cells are used in assessing one or moretest compounds.

In some embodiments, one or more vectors described herein are used toproduce a non-human transgenic animal or transgenic plant. In someembodiments, the transgenic animal is a mammal, such as a mouse, rat, orrabbit. Methods for producing transgenic plants and animals are known inthe art, and generally begin with a method of cell transfection, such asdescribed herein.

With recent advances in crop genomics, the ability to use CRISPR-Cassystems to perform efficient and cost effective gene editing andmanipulation will allow the rapid selection and comparison of single andand multiplexed genetic manipulations to transform such genomes forimproved production and enhanced traits. In this regard reference ismade to US patents and publications: U.S. Pat. No.6,603,061—Agrobacterium-Mediated Plant Transformation Method; U.S. Pat.No. 7,868,149—Plant Genome Sequences and Uses Thereof and US2009/0100536—Transgenic Plants with Enhanced Agronomic Traits, all thecontents and disclosure of each of which are herein incorporated byreference in their entirety. In the practice of the invention, thecontents and disclosure of Morrell et al “Crop genomics: advances andapplications” Nat Rev Genet. 2011 Dec. 29; 13(2):85-96 are also hereinincorporated by reference in their entirety. In an advantageousembodiment of the invention, the CRISPR/Cas9 system is used to engineermicroalgae. That the CRISPR-Cas system is able to be employed in plantsystems is also provided in the manuscript “Efficient Genome Editing inPlants using a CRISPR/Cas System”, by Feng et al. Cell Res. 2013 Aug.20. doi: 10.1038/cr.2013.114. [Epub ahead of print], incorporated byreference in its entirety, wherein it is demonstrated that engineeredCRISPR/Cas complexes may be used to create double strand breaks atspecific sites of the plant genome to achieve targeted genomemodifications in both dicot and monocot plants. Accordingly, referenceherein to animal cells may also apply, mutatis mutandis, to plant cellsunless otherwise apparent.

In one aspect, the invention provides for methods of modifying a targetpolynucleotide in a eukaryotic cell, which may be in vivo, ex vivo or invitro. In some embodiments, the method comprises sampling a cell orpopulation of cells from a human or non-human animal or plant (includingmicro-algae), and modifying the cell or cells. Culturing may occur atany stage ex vivo. The cell or cells may even be re-introduced into thenon-human animal or plant (including micro-algae). For re-introducedcells it is particularly preferred that the cells are stem cells.

In some embodiments, the method comprises allowing a CRISPR complex tobind to the target polynucleotide to effect cleavage of said targetpolynucleotide thereby modifying the target polynucleotide, wherein theCRISPR complex comprises a CRISPR enzyme complexed with a guide sequencehybridized to a target sequence within said target polynucleotide,wherein said guide sequence is linked to a tracr mate sequence which inturn hybridizes to a tracr sequence.

In one aspect, the invention provides a method of modifying expressionof a polynucleotide in a eukaryotic cell. In some embodiments, themethod comprises allowing a CRISPR complex to bind to the polynucleotidesuch that said binding results in increased or decreased expression ofsaid polynucleotide; wherein the CRISPR complex comprises a CRISPRenzyme complexed with a guide sequence hybridized to a target sequencewithin said polynucleotide, wherein said guide sequence is linked to atracr mate sequence which in turn hybridizes to a tracr sequence.Similar considerations and conditions apply as above for methods ofmodifying a target polynucleotide. In fact, these sampling, culturingand re-introduction options apply across the aspects of the presentinvention.

Indeed, in any aspect of the invention, the CRISPR complex may comprisea CRISPR enzyme complexed with a guide sequence hybridized to a targetsequence, wherein said guide sequence may be linked to a tracr matesequence which in turn may hybridize to a tracr sequence. Similarconsiderations and conditions apply as above for methods of modifying atarget polynucleotide.

In one aspect, the invention provides kits containing any one or more ofthe elements disclosed in the above methods and compositions. Elementsmay be provided individually or in combinations, and may be provided inany suitable container, such as a vial, a bottle, or a tube. In someembodiments, the kit includes instructions in one or more languages, forexample in more than one language.

In some embodiments, a kit comprises one or more reagents for use in aprocess utilizing one or more of the elements described herein. Reagentsmay be provided in any suitable container. For example, a kit mayprovide one or more reaction or storage buffers. Reagents may beprovided in a form that is usable in a particular assay, or in a formthat requires addition of one or more other components before use (e.g.in concentrate or lyophilized form). A buffer can be any buffer,including but not limited to a sodium carbonate buffer, a sodiumbicarbonate buffer, a borate buffer, a Tris buffer, a MOPS buffer, aHEPES buffer, and combinations thereof. In some embodiments, the bufferis alkaline. In some embodiments, the buffer has a pH from about 7 toabout 10. In some embodiments, the kit comprises one or moreoligonucleotides corresponding to a guide sequence for insertion into avector so as to operably link the guide sequence and a regulatoryelement. In some embodiments, the kit comprises a homologousrecombination template polynucleotide.

In one aspect, the invention provides methods for using one or moreelements of a CRISPR system. The CRISPR complex of the inventionprovides an effective means for modifying a target polynucleotide. TheCRISPR complex of the invention has a wide variety of utility includingmodifying (e.g., deleting, inserting, translocating, inactivating,activating) a target polynucleotide in a multiplicity of cell types. Assuch the CRISPR complex of the invention has a broad spectrum ofapplications in, e.g., gene therapy, drug screening, disease diagnosis,and prognosis. An exemplary CRISPR complex comprises a CRISPR enzymecomplexed with a guide sequence hybridized to a target sequence withinthe target polynucleotide. The guide sequence is linked to a tracr matesequence, which in turn hybridizes to a tracr sequence.

In one embodiment, this invention provides a method of cleaving a targetpolynucleotide. The method comprises modifying a target polynucleotideusing a CRISPR complex that binds to the target polynucleotide andeffect cleavage of said target polynucleotide. Typically, the CRISPRcomplex of the invention, when introduced into a cell, creates a break(e.g., a single or a double strand break) in the genome sequence. Forexample, the method can be used to cleave a disease gene in a cell.

The break created by the CRISPR complex can be repaired by a repairprocesses such as the error prone non-homologous end joining (NHEJ)pathway or the high fidelity homology-directed repair (HDR) (FIG. 11).During these repair process, an exogenous polynucleotide template can beintroduced into the genome sequence. In some methods, the HDR process isused modify genome sequence. For example, an exogenous polynucleotidetemplate comprising a sequence to be integrated flanked by an upstreamsequence and a downstream sequence is introduced into a cell. Theupstream and downstream sequences share sequence similarity with eitherside of the site of integration in the chromosome.

Where desired, a donor polynucleotide can be DNA, e.g., a DNA plasmid, abacterial artificial chromosome (BAC), a yeast artificial chromosome(YAC), a viral vector, a linear piece of DNA, a PCR fragment, a nakednucleic acid, or a nucleic acid complexed with a delivery vehicle suchas a liposome or poloxamer.

The exogenous polynucleotide template comprises a sequence to beintegrated (e.g., a mutated gene). The sequence for integration may be asequence endogenous or exogenous to the cell. Examples of a sequence tobe integrated include polynucleotides encoding a protein or a non-codingRNA (e.g., a microRNA). Thus, the sequence for integration may beoperably linked to an appropriate control sequence or sequences.Alternatively, the sequence to be integrated may provide a regulatoryfunction.

The upstream and downstream sequences in the exogenous polynucleotidetemplate are selected to promote recombination between the chromosomalsequence of interest and the donor polynucleotide. The upstream sequenceis a nucleic acid sequence that shares sequence similarity with thegenome sequence upstream of the targeted site for integration.Similarly, the downstream sequence is a nucleic acid sequence thatshares sequence similarity with the chromosomal sequence downstream ofthe targeted site of integration. The upstream and downstream sequencesin the exogenous polynucleotide template can have 75%, 80%, 85%, 90%,95%, or 100% sequence identity with the targeted genome sequence.Preferably, the upstream and downstream sequences in the exogenouspolynucleotide template have about 95%, 96%, 97%, 98%, 99%, or 100%sequence identity with the targeted genome sequence. In some methods,the upstream and downstream sequences in the exogenous polynucleotidetemplate have about 99% or 100% sequence identity with the targetedgenome sequence.

An upstream or downstream sequence may comprise from about 20 bp toabout 2500 bp, for example, about 50, 100, 200, 300, 400, 500, 600, 700,800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900,2000, 2100, 2200, 2300, 2400, or 2500 bp. In some methods, the exemplaryupstream or downstream sequence have about 200 bp to about 2000 bp,about 600 bp to about 1000 bp, or more particularly about 700 bp toabout 1000 bp.

In some methods, the exogenous polynucleotide template may furthercomprise a marker. Such a marker may make it easy to screen for targetedintegrations. Examples of suitable markers include restriction sites,fluorescent proteins, or selectable markers. The exogenouspolynucleotide template of the invention can be constructed usingrecombinant techniques (see, for example, Sambrook et al., 2001 andAusubel et al., 1996).

In an exemplary method for modifying a target polynucleotide byintegrating an exogenous polynucleotide template, a double strandedbreak is introduced into the genome sequence by the CRISPR complex, thebreak is repaired via homologous recombination an exogenouspolynucleotide template such that the template is integrated into thegenome. The presence of a double-stranded break facilitates integrationof the template.

In other embodiments, this invention provides a method of modifyingexpression of a polynucleotide in a eukaryotic cell. The methodcomprises increasing or decreasing expression of a target polynucleotideby using a CRISPR complex that binds to the polynucleotide.

In some methods, a target polynucleotide can be inactivated to effectthe modification of the expression in a cell. For example, upon thebinding of a CRISPR complex to a target sequence in a cell, the targetpolynucleotide is inactivated such that the sequence is not transcribed,the coded protein is not produced, or the sequence does not function asthe wild-type sequence does. For example, a protein or microRNA codingsequence may be inactivated such that the protein is not produced.

In some methods, a control sequence can be inactivated such that it nolonger functions as a control sequence. As used herein, “controlsequence” refers to any nucleic acid sequence that effects thetranscription, translation, or accessibility of a nucleic acid sequence.Examples of a control sequence include, a promoter, a transcriptionterminator, and an enhancer are control sequences.

The inactivated target sequence may include a deletion mutation (i.e.,deletion of one or more nucleotides), an insertion mutation (i.e.,insertion of one or more nucleotides), or a nonsense mutation (i.e.,substitution of a single nucleotide for another nucleotide such that astop codon is introduced). In some methods, the inactivation of a targetsequence results in “knock-out” of the target sequence.

A method of the invention may be used to create a plant, an animal orcell that may be used as a disease model. As used herein, “disease”refers to a disease, disorder, or indication in a subject. For example,a method of the invention may be used to create an animal or cell thatcomprises a modification in one or more nucleic acid sequencesassociated with a disease, or a plant, animal or cell in which theexpression of one or more nucleic acid sequences associated with adisease are altered. Such a nucleic acid sequence may encode a diseaseassociated protein sequence or may be a disease associated controlsequence. Accordingly, it is understood that in embodiments of theinvention, a plant, subject, patient, organism or cell can be anon-human subject, patient, organism or cell. Thus, the inventionprovides a plant, animal or cell, produced by the present methods, or aprogeny thereof. The progeny may be a clone of the produced plant oranimal, or may result from sexual reproduction by crossing with otherindividuals of the same species to introgress further desirable traitsinto their offspring. The cell may be in vivo or ex vivo in the cases ofmulticellular organisms, particularly animals or plants. In the instancewhere the cell is in cultured, a cell line may be established ifappropriate culturing conditions are met and preferably if the cell issuitably adapted for this purpose (for instance a stem cell). Bacterialcell lines produced by the invention are also envisaged. Hence, celllines are also envisaged.

In some methods, the disease model can be used to study the effects ofmutations on the animal or cell and development and/or progression ofthe disease using measures commonly used in the study of the disease.Alternatively, such a disease model is useful for studying the effect ofa pharmaceutically active compound on the disease.

In some methods, the disease model can be used to assess the efficacy ofa potential gene therapy strategy. That is, a disease-associated gene orpolynucleotide can be modified such that the disease development and/orprogression is inhibited or reduced. In particular, the method comprisesmodifying a disease-associated gene or polynucleotide such that analtered protein is produced and, as a result, the animal or cell has analtered response. Accordingly, in some methods, a genetically modifiedanimal may be compared with an animal predisposed to development of thedisease such that the effect of the gene therapy event may be assessed.

In another embodiment, this invention provides a method of developing abiologically active agent that modulates a cell signaling eventassociated with a disease gene. The method comprises contacting a testcompound with a cell comprising one or more vectors that driveexpression of one or more of a CRISPR enzyme, a guide sequence linked toa tracr mate sequence, and a tracr sequence; and detecting a change in areadout that is indicative of a reduction or an augmentation of a cellsignaling event associated with, e.g., a mutation in a disease genecontained in the cell.

A cell model or animal model can be constructed in combination with themethod of the invention for screening a cellular function change. Such amodel may be used to study the effects of a genome sequence modified bythe CRISPR complex of the invention on a cellular function of interest.For example, a cellular function model may be used to study the effectof a modified genome sequence on intracellular signaling orextracellular signaling. Alternatively, a cellular function model may beused to study the effects of a modified genome sequence on sensoryperception. In some such models, one or more genome sequences associatedwith a signaling biochemical pathway in the model are modified.

Several disease models have been specifically investigated. Theseinclude de novo autism risk genes CHD8, KATNAL2, and SCN2A; and thesyndromic autism (Angelman Syndrome) gene UBE3A. These genes andresulting autism models are of course preferred, but serve to show thebroad applicability of the invention across genes and correspondingmodels.

An altered expression of one or more genome sequences associated with asignaling biochemical pathway can be determined by assaying for adifference in the mRNA levels of the corresponding genes between thetest model cell and a control cell, when they are contacted with acandidate agent. Alternatively, the differential expression of thesequences associated with a signaling biochemical pathway is determinedby detecting a difference in the level of the encoded polypeptide orgene product.

To assay for an agent-induced alteration in the level of mRNAtranscripts or corresponding polynucleotides, nucleic acid contained ina sample is first extracted according to standard methods in the art.For instance, mRNA can be isolated using various lytic enzymes orchemical solutions according to the procedures set forth in Sambrook etal. (1989), or extracted by nucleic-acid-binding resins following theaccompanying instructions provided by the manufacturers. The mRNAcontained in the extracted nucleic acid sample is then detected byamplification procedures or conventional hybridization assays (e.g.Northern blot analysis) according to methods widely known in the art orbased on the methods exemplified herein.

For purpose of this invention, amplification means any method employinga primer and a polymerase capable of replicating a target sequence withreasonable fidelity. Amplification may be carried out by natural orrecombinant DNA polymerases such as TaqGoldTm, T7 DNA polymerase, Klenowfragment of E. coli DNA polymerase, and reverse transcriptase. Apreferred amplification method is PCR. In particular, the isolated RNAcan be subjected to a reverse transcription assay that is coupled with aquantitative polymerase chain reaction (RT-PCR) in order to quantify theexpression level of a sequence associated with a signaling biochemicalpathway.

Detection of the gene expression level can be conducted in real time inan amplification assay. In one aspect, the amplified products can bedirectly visualized with fluorescent DNA-binding agents including butnot limited to DNA intercalators and DNA groove binders. Because theamount of the intercalators incorporated into the double-stranded DNAmolecules is typically proportional to the amount of the amplified DNAproducts, one can conveniently determine the amount of the amplifiedproducts by quantifying the fluorescence of the intercalated dye usingconventional optical systems in the art. DNA-binding dye suitable forthis application include SYBR green, SYBR blue, DAPI, propidium iodine,Hoeste, SYBR gold, ethidium bromide, acridines, proflavine, acridineorange, acriflavine, fluorcoumanin, ellipticine, daunomycin,chloroquine, distamycin D, chromomycin, homidium, mithramycin, rutheniumpolypyridyls, anthramycin, and the like.

In another aspect, other fluorescent labels such as sequence specificprobes can be employed in the amplification reaction to facilitate thedetection and quantification of the amplified products. Probe-basedquantitative amplification relies on the sequence-specific detection ofa desired amplified product. It utilizes fluorescent, target-specificprobes (e.g., TaqMan® probes) resulting in increased specificity andsensitivity. Methods for performing probe-based quantitativeamplification are well established in the art and are taught in U.S.Pat. No. 5,210,015.

In yet another aspect, conventional hybridization assays usinghybridization probes that share sequence homology with sequencesassociated with a signaling biochemical pathway can be performed.Typically, probes are allowed to form stable complexes with thesequences associated with a signaling biochemical pathway containedwithin the biological sample derived from the test subject in ahybridization reaction. It will be appreciated by one of skill in theart that where antisense is used as the probe nucleic acid, the targetpolynucleotides provided in the sample are chosen to be complementary tosequences of the antisense nucleic acids. Conversely, where thenucleotide probe is a sense nucleic acid, the target polynucleotide isselected to be complementary to sequences of the sense nucleic acid.

Hybridization can be performed under conditions of various stringency.Suitable hybridization conditions for the practice of the presentinvention are such that the recognition interaction between the probeand sequences associated with a signaling biochemical pathway is bothsufficiently specific and sufficiently stable. Conditions that increasethe stringency of a hybridization reaction are widely known andpublished in the art. See, for example, (Sambrook, et al., (1989);Nonradioactive In Situ Hybridization Application Manual, BoehringerMannheim, second edition). The hybridization assay can be formed usingprobes immobilized on any solid support, including but are not limitedto nitrocellulose, glass, silicon, and a variety of gene arrays. Apreferred hybridization assay is conducted on high-density gene chips asdescribed in U.S. Pat. No. 5,445,934.

For a convenient detection of the probe-target complexes formed duringthe hybridization assay, the nucleotide probes are conjugated to adetectable label. Detectable labels suitable for use in the presentinvention include any composition detectable by photochemical,biochemical, spectroscopic, immunochemical, electrical, optical orchemical means. A wide variety of appropriate detectable labels areknown in the art, which include fluorescent or chemiluminescent labels,radioactive isotope labels, enzymatic or other ligands. In preferredembodiments, one will likely desire to employ a fluorescent label or anenzyme tag, such as digoxigenin, β-galactosidase, urease, alkalinephosphatase or peroxidase, avidin/biotin complex.

The detection methods used to detect or quantify the hybridizationintensity will typically depend upon the label selected above. Forexample, radiolabels may be detected using photographic film or aphosphoimager. Fluorescent markers may be detected and quantified usinga photodetector to detect emitted light. Enzymatic labels are typicallydetected by providing the enzyme with a substrate and measuring thereaction product produced by the action of the enzyme on the substrate;and finally colorimetric labels are detected by simply visualizing thecolored label.

An agent-induced change in expression of sequences associated with asignaling biochemical pathway can also be determined by examining thecorresponding gene products. Determining the protein level typicallyinvolves a) contacting the protein contained in a biological sample withan agent that specifically bind to a protein associated with a signalingbiochemical pathway; and (b) identifying any agent:protein complex soformed. In one aspect of this embodiment, the agent that specificallybinds a protein associated with a signaling biochemical pathway is anantibody, preferably a monoclonal antibody.

The reaction is performed by contacting the agent with a sample of theproteins associated with a signaling biochemical pathway derived fromthe test samples under conditions that will allow a complex to formbetween the agent and the proteins associated with a signalingbiochemical pathway. The formation of the complex can be detecteddirectly or indirectly according to standard procedures in the art. Inthe direct detection method, the agents are supplied with a detectablelabel and unreacted agents may be removed from the complex; the amountof remaining label thereby indicating the amount of complex formed. Forsuch method, it is preferable to select labels that remain attached tothe agents even during stringent washing conditions. It is preferablethat the label does not interfere with the binding reaction. In thealternative, an indirect detection procedure may use an agent thatcontains a label introduced either chemically or enzymatically. Adesirable label generally does not interfere with binding or thestability of the resulting agent:polypeptide complex. However, the labelis typically designed to be accessible to an antibody for an effectivebinding and hence generating a detectable signal.

A wide variety of labels suitable for detecting protein levels are knownin the art. Non-limiting examples include radioisotopes, enzymes,colloidal metals, fluorescent compounds, bioluminescent compounds, andchemiluminescent compounds.

The amount of agent:polypeptide complexes formed during the bindingreaction can be quantified by standard quantitative assays. Asillustrated above, the formation of agent:polypeptide complex can bemeasured directly by the amount of label remained at the site ofbinding. In an alternative, the protein associated with a signalingbiochemical pathway is tested for its ability to compete with a labeledanalog for binding sites on the specific agent. In this competitiveassay, the amount of label captured is inversely proportional to theamount of protein sequences associated with a signaling biochemicalpathway present in a test sample.

A number of techniques for protein analysis based on the generalprinciples outlined above are available in the art. They include but arenot limited to radioimmunoassays, ELISA (enzyme linked immunoradiometricassays), “sandwich” immunoassays, immunoradiometric assays, in situimmunoassays (using e.g., colloidal gold, enzyme or radioisotopelabels), western blot analysis, immunoprecipitation assays,immunofluorescent assays, and SDS-PAGE.

Antibodies that specifically recognize or bind to proteins associatedwith a signaling biochemical pathway are preferable for conducting theaforementioned protein analyses. Where desired, antibodies thatrecognize a specific type of post-translational modifications (e.g.,signaling biochemical pathway inducible modifications) can be used.Post-translational modifications include but are not limited toglycosylation, lipidation, acetylation, and phosphorylation. Theseantibodies may be purchased from commercial vendors. For example,anti-phosphotyrosine antibodies that specifically recognizetyrosine-phosphorylated proteins are available from a number of vendorsincluding Invitrogen and Perkin Elmer. Anti-phosphotyrosine antibodiesare particularly useful in detecting proteins that are differentiallyphosphorylated on their tyrosine residues in response to an ER stress.Such proteins include but are not limited to eukaryotic translationinitiation factor 2 alpha (eIF-2α). Alternatively, these antibodies canbe generated using conventional polyclonal or monoclonal antibodytechnologies by immunizing a host animal or an antibody-producing cellwith a target protein that exhibits the desired post-translationalmodification.

In practicing the subject method, it may be desirable to discern theexpression pattern of an protein associated with a signaling biochemicalpathway in different bodily tissue, in different cell types, and/or indifferent subcellular structures. These studies can be performed withthe use of tissue-specific, cell-specific or subcellular structurespecific antibodies capable of binding to protein markers that arepreferentially expressed in certain tissues, cell types, or sub cellularstructures.

An altered expression of a gene associated with a signaling biochemicalpathway can also be determined by examining a change in activity of thegene product relative to a control cell. The assay for an agent-inducedchange in the activity of a protein associated with a signalingbiochemical pathway will dependent on the biological activity and/or thesignal transduction pathway that is under investigation. For example,where the protein is a kinase, a change in its ability to phosphorylatethe downstream substrate(s) can be determined by a variety of assaysknown in the art. Representative assays include but are not limited toimmunoblotting and immunoprecipitation with antibodies such asanti-phosphotyrosine antibodies that recognize phosphorylated proteins.In addition, kinase activity can be detected by high throughputchemiluminescent assays such as AlphaScreen™ (available from PerkinElmer) and eTag™ assay (Chan-Hui, et al. (2003) Clinical Immunology 111:162-174).

Where the protein associated with a signaling biochemical pathway ispart of a signaling cascade leading to a fluctuation of intracellular pHcondition, pH sensitive molecules such as fluorescent pH dyes can beused as the reporter molecules. In another example where the proteinassociated with a signaling biochemical pathway is an ion channel,fluctuations in membrane potential and/or intracellular ionconcentration can be monitored. A number of commercial kits andhigh-throughput devices are particularly suited for a rapid and robustscreening for modulators of ion channels. Representative instrumentsinclude FLIPR™ (Molecular Devices, Inc.) and VIPR (Aurora Biosciences).These instruments are capable of detecting reactions in over 1000 samplewells of a microplate simultaneously, and providing real-timemeasurement and functional data within a second or even a minisecond.

In practicing any of the methods disclosed herein, a suitable vector canbe introduced to a cell or an embryo via one or more methods known inthe art, including without limitation, microinjection, electroporation,sonoporation, biolistics, calcium phosphate-mediated transfection,cationic transfection, liposome transfection, dendrimer transfection,heat shock transfection, nucleofection transfection, magnetofection,lipofection, impalefection, optical transfection, proprietaryagent-enhanced uptake of nucleic acids, and delivery via liposomes,immunoliposomes, virosomes, or artificial virions. In some methods, thevector is introduced into an embryo by microinjection. The vector orvectors may be microinjected into the nucleus or the cytoplasm of theembryo. In some methods, the vector or vectors may be introduced into acell by nucleofection.

The target polynucleotide of a CRISPR complex can be any polynucleotideendogenous or exogenous to the eukaryotic cell. For example, the targetpolynucleotide can be a polynucleotide residing in the nucleus of theeukaryotic cell. The target polynucleotide can be a sequence coding agene product (e.g., a protein) or a non-coding sequence (e.g., aregulatory polynucleotide or a junk DNA).

Examples of target polynucleotides include a sequence associated with asignaling biochemical pathway, e.g., a signaling biochemicalpathway-associated gene or polynucleotide. Examples of targetpolynucleotides include a disease associated gene or polynucleotide. A“disease-associated” gene or polynucleotide refers to any gene orpolynucleotide which is yielding transcription or translation productsat an abnormal level or in an abnormal form in cells derived from adisease-affected tissues compared with tissues or cells of a non diseasecontrol. It may be a gene that becomes expressed at an abnormally highlevel; it may be a gene that becomes expressed at an abnormally lowlevel, where the altered expression correlates with the occurrenceand/or progression of the disease. A disease-associated gene also refersto a gene possessing mutation(s) or genetic variation that is directlyresponsible or is in linkage disequilibrium with a gene(s) that isresponsible for the etiology of a disease. The transcribed or translatedproducts may be known or unknown, and may be at a normal or abnormallevel.

The target polynucleotide of a CRISPR complex can be any polynucleotideendogenous or exogenous to the eukaryotic cell. For example, the targetpolynucleotide can be a polynucleotide residing in the nucleus of theeukaryotic cell. The target polynucleotide can be a sequence coding agene product (e.g., a protein) or a non-coding sequence (e.g., aregulatory polynucleotide or a junk DNA).

The target polynucleotide of a CRISPR complex may include a number ofdisease-associated genes and polynucleotides as well as signalingbiochemical pathway-associated genes and polynucleotides as listed inU.S. provisional patent applications 61/736,527 and 61/748,427 havingBroad reference BI-2011/008/WSGR Docket No. 44063-701.101 andBI-2011/008/WSGR Docket No. 44063-701.102 respectively, both entitledSYSTEMS METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION filed on Dec.12, 2012 and Jan. 2, 2013, respectively, the contents of all of whichare herein incorporated by reference in their entirety.

Examples of target polynucleotides include a sequence associated with asignaling biochemical pathway, e.g., a signaling biochemicalpathway-associated gene or polynucleotide. Examples of targetpolynucleotides include a disease associated gene or polynucleotide. A“disease-associated” gene or polynucleotide refers to any gene orpolynucleotide which is yielding transcription or translation productsat an abnormal level or in an abnormal form in cells derived from adisease-affected tissues compared with tissues or cells of a non diseasecontrol. It may be a gene that becomes expressed at an abnormally highlevel; it may be a gene that becomes expressed at an abnormally lowlevel, where the altered expression correlates with the occurrenceand/or progression of the disease. A disease-associated gene also refersto a gene possessing mutation(s) or genetic variation that is directlyresponsible or is in linkage disequilibrium with a gene(s) that isresponsible for the etiology of a disease. The transcribed or translatedproducts may be known or unknown, and may be at a normal or abnormallevel.

Examples of disease-associated genes and polynucleotides are listed inTables A and B. Disease specific information is available fromMcKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University(Baltimore, Md.) and National Center for Biotechnology Information,National Library of Medicine (Bethesda, Md.), available on the WorldWide Web. Examples of signaling biochemical pathway-associated genes andpolynucleotides are listed in Table C.

Mutations in these genes and pathways can result in production ofimproper proteins or proteins in improper amounts which affect function.Further examples of genes, diseases and proteins are hereby incorporatedby reference from U.S. Provisional applications 61/736,527 filed on Dec.12, 2012 and 61/748,427 filed on Jan. 2, 2013. Such genes, proteins andpathways may be the target polynucleotide of a CRISPR complex.

TABLE A DISEASE/DISORDERS GENE(S) Neoplasia PTEN; ATM; ATR; EGFR; ERBB2;ERBB3; ERBB4; Notch1; Notch2; Notch3; Notch4; AKT; AKT2; AKT3; HIF;HIF1a; HIF3a; Met; HRG; Bcl2; PPAR alpha; PPAR gamma; WT1 (Wilms Tumor);FGF Receptor Family members (5 members: 1, 2, 3, 4, 5); CDKN2a; APC; RB(retinoblastoma); MEN1; VHL; BRCA1; BRCA2; AR (Androgen Receptor);TSG101; IGF; IGF Receptor; Igf1 (4 variants); Igf2 (3 variants); Igf 1Receptor; Igf 2 Receptor; Bax; Bcl2; caspases family (9 members: 1, 2,3, 4, 6, 7, 8, 9, 12); Kras; Apc Age-related Macular Abcr; Ccl2; Cc2; cp(ceruloplasmin); Timp3; cathepsinD; Degeneration Vldlr; Ccr2Schizophrenia Neuregulin1 (Nrg1); Erb4 (receptor for Neuregulin);Complexin1 (Cplx1); Tph1 Tryptophan hydroxylase; Tph2 Tryptophanhydroxylase 2; Neurexin 1; GSK3; GSK3a; GSK3b Disorders 5-HTT (Slc6a4);COMT; DRD (Drd1a); SLC6A3; DAOA; DTNBP1; Dao (Dao1) Trinucleotide HTT(Huntington's Dx); SBMA/SMAX1/AR (Kennedy's Repeat Disorders Dx);FXN/X25 (Friedrich's Ataxia); ATX3 (Machado- Joseph's Dx); ATXN1 andATXN2 (spinocerebellar ataxias); DMPK (myotonic dystrophy); Atrophin-1and Atn1 (DRPLA Dx); CBP (Creb-BP - global instability); VLDLR(Alzheimer's); Atxn7; Atxn10 Fragile X Syndrome FMR2; FXR1; FXR2; mGLUR5Secretase Related APH-1 (alpha and beta); Presenilin (Psen1); nicastrinDisorders (Ncstn); PEN-2 Others Nos1; Parp1; Nat1; Nat2 Prion - relateddisorders Prp ALS SOD1; ALS2; STEX; FUS; TARDBP; VEGF (VEGF-a; VEGF-b;VEGF-c) Drug addiction Prkce (alcohol); Drd2; Drd4; ABAT (alcohol);GRIA2; Grm5; Grin1; Htr1b; Grin2a; Drd3; Pdyn; Gria1 (alcohol) AutismMecp2; BZRAP1; MDGA2; Sema5A; Neurexin 1; Fragile X (FMR2 (AFF2); FXR1;FXR2; Mglur5) Alzheimer's Disease E1; CHIP; UCH; UBB; Tau; LRP; PICALM;Clusterin; PS1; SORL1; CR1; Vldlr; Uba1; Uba3; CHIP28 (Aqp1, Aquaporin1); Uchl1; Uchl3; APP Inflammation IL-10; IL-1 (IL-1a; IL-1b); IL-13;IL-17 (IL-17a (CTLA8); IL- 17b; IL-17c; IL-17d; IL-17f); II-23; Cx3cr1;ptpn22; TNFa; NOD2/CARD15 for IBD; IL-6; IL-12 (IL-12a; IL-12b); CTLA4;Cx3cl1 Parkinson's Disease x-Synuclein; DJ-1; LRRK2; Parkin; PINK1

TABLE B Blood and Anemia (CDAN1, CDA1, RPS19, DBA, PKLR, PK1, NT5C3,UMPH1, coagulation diseases PSN1, RHAG, RH50A, NRAMP2, SPTB, ALAS2,ANH1, ASB, and disorders ABCB7, ABC7, ASAT); Bare lymphocyte syndrome(TAPBP, TPSN, TAP2, ABCB3, PSF2, RING11, MHC2TA, C2TA, RFX5, RFXAP,RFX5), Bleeding disorders (TBXA2R, P2RX1, P2X1); Factor H and factorH-like 1 (HF1, CFH, HUS); Factor V and factor VIII (MCFD2); Factor VIIdeficiency (F7); Factor X deficiency (F10); Factor XI deficiency (F11);Factor XII deficiency (F12, HAF); Factor XIIIA deficiency (F13A1, F13A);Factor XIIIB deficiency (F13B); Fanconi anemia (FANCA, FACA, FA1, FA,FAA, FAAP95, FAAP90, FLJ34064, FANCB, FANCC, FACC, BRCA2, FANCD1,FANCD2, FANCD, FACD, FAD, FANCE, FACE, FANCF, XRCC9, FANCG, BRIP1,BACH1, FANCJ, PHF9, FANCL, FANCM, KIAA1596); Hemophagocyticlymphohistiocytosis disorders (PRF1, HPLH2, UNC13D, MUNC13-4, HPLH3,HLH3, FHL3); Hemophilia A (F8, F8C, HEMA); Hemophilia B (F9, HEMB),Hemorrhagic disorders (PI, ATT, F5); Leukocyde deficiencies anddisorders (ITGB2, CD18, LCAMB, LAD, EIF2B1, EIF2BA, EIF2B2, EIF2B3,EIF2B5, LVWM, CACH, CLE, EIF2B4); Sickle cell anemia (HBB); Thalassemia(HBA2, HBB, HBD, LCRB, HBA1). Cell dysregulation B-cell non-Hodgkinlymphoma (BCL7A, BCL7); Leukemia (TAL1 and oncology TCL5, SCL, TAL2,FLT3, NBS1, NBS, ZNFN1A1, IK1, LYF1, diseases and disorders HOXD4,HOX4B, BCR, CML, PHL, ALL, ARNT, KRAS2, RASK2, GMPS, AF10, ARHGEF12,LARG, KIAA0382, CALM, CLTH, CEBPA, CEBP, CHIC2, BTL, FLT3, KIT, PBT,LPP, NPM1, NUP214, D9S46E, CAN, CAIN, RUNX1, CBFA2, AML1, WHSC1L1, NSD3,FLT3, AF1Q, NPM1, NUMA1, ZNF145, PLZF, PML, MYL, STAT5B, AF10, CALM,CLTH, ARL11, ARLTS1, P2RX7, P2X7, BCR, CML, PHL, ALL, GRAF, NF1, VRNF,WSS, NFNS, PTPN11, PTP2C, SHP2, NS1, BCL2, CCND1, PRAD1, BCL1, TCRA,GATA1, GF1, ERYF1, NFE1, ABL1, NQO1, DIA4, NMOR1, NUP214, D9S46E, CAN,CAIN). Inflammation and AIDS (KIR3DL1, NKAT3, NKB1, AMB11, KIR3DS1,IFNG, CXCL12, immune related SDF1); Autoimmune lymphoproliferativesyndrome (TNFRSF6, APT1, diseases and disorders FAS, CD95, ALPS1A);Combined immunodeficiency, (IL2RG, SCIDX1, SCIDX, IMD4); HIV-1 (CCL5,SCYA5, D17S136E, TCP228), HIV susceptibility or infection (IL10, CSIF,CMKBR2, CCR2, CMKBR5, CCCKR5 (CCR5)); Immunodeficiencies (CD3E, CD3G,AICDA, AID, HIGM2, TNFRSF5, CD40, UNG, DGU, HIGM4, TNFSF5, CD40LG,HIGM1, IGM, FOXP3, IPEX, AIID, XPID, PIDX, TNFRSF14B, TACI);Inflammation (IL-10, IL-1 (IL-1a, IL-1b), IL-13, IL-17 (IL-17a (CTLA8),IL-17b, IL-17c, IL-17d, IL-17f), II-23, Cx3cr1, ptpn22, TNFa,NOD2/CARD15 for IBD, IL-6, IL-12 (IL-12a, IL-12b), CTLA4, Cx3cl1);Severe combined immunodeficiencies (SCIDs)(JAK3, JAKL, DCLRE1C, ARTEMIS,SCIDA, RAG1, RAG2, ADA, PTPRC, CD45, LCA, IL7R, CD3D, T3D, IL2RG,SCIDX1, SCIDX, IMD4). Metabolic, liver, Amyloid neuropathy (TTR, PALB);Amyloidosis (APOA1, APP, AAA, kidney and protein CVAP, AD1, GSN, FGA,LYZ, TTR, PALB); Cirrhosis (KRT18, KRT8, diseases and disorders CIRH1A,NAIC, TEX292, KIAA1988); Cystic fibrosis (CFTR, ABCC7, CF, MRP7);Glycogen storage diseases (SLC2A2, GLUT2, G6PC, G6PT, G6PT1, GAA, LAMP2,LAMPB, AGL, GDE, GBE1, GYS2, PYGL, PFKM); Hepatic adenoma, 142330 (TCF1,HNF1A, MODY3), Hepatic failure, early onset, and neurologic disorder(SCOD1, SCO1), Hepatic lipase deficiency (LIPC), Hepatoblastoma, cancerand carcinomas (CTNNB1, PDGFRL, PDGRL, PRLTS, AXIN1, AXIN, CTNNB1, TP53,P53, LFS1, IGF2R, MPRI, MET, CASP8, MCH5; Medullary cystic kidneydisease (UMOD, HNFJ, FJHN, MCKD2, ADMCKD2); Phenylketonuria (PAH, PKU1,QDPR, DHPR, PTS); Polycystic kidney and hepatic disease (FCYT, PKHD1,ARPKD, PKD1, PKD2, PKD4, PKDTS, PRKCSH, G19P1, PCLD, SEC63).Muscular/Skeletal Becker muscular dystrophy (DMD, BMD, MYF6), DuchenneMuscular diseases and disorders Dystrophy (DMD, BMD); Emery-Dreifussmuscular dystrophy (LMNA, LMN1, EMD2, FPLD, CMD1A, HGPS, LGMD1B, LMNA,LMN1, EMD2, FPLD, CMD1A); Facioscapulohumeral muscular dystrophy(FSHMD1A, FSHD1A); Muscular dystrophy (FKRP, MDC1C, LGMD2I, LAMA2, LAMM,LARGE, KIAA0609, MDC1D, FCMD, TTID, MYOT, CAPN3, CANP3, DYSF, LGMD2B,SGCG, LGMD2C, DMDA1, SCG3, SGCA, ADL, DAG2, LGMD2D, DMDA2, SGCB, LGMD2E,SGCD, SGD, LGMD2F, CMD1L, TCAP, LGMD2G, CMD1N, TRIM32, HT2A, LGMD2H,FKRP, MDC1C, LGMD2I, TTN, CMD1G, TMD, LGMD2J, POMT1, CAV3, LGMD1C,SEPN1, SELN, RSMD1, PLEC1, PLTN, EBS1); Osteopetrosis (LRP5, BMND1,LRP7, LR3, OPPG, VBCH2, CLCN7, CLC7, OPTA2, OSTM1, GL, TCIRG1, TIRC7,OC116, OPTB1); Muscular atrophy (VAPB, VAPC, ALS8, SMN1, SMA1, SMA2,SMA3, SMA4, BSCL2, SPG17, GARS, SMAD1, CMT2D, HEXB, IGHMBP2, SMUBP2,CATF1, SMARD1). Neurological and ALS (SOD1, ALS2, STEX, FUS, TARDBP,VEGF (VEGF-a, VEGF-b, neuronal diseases VEGF-c); Alzheimer disease (APP,AAA, CVAP, AD1, APOE, AD2, and disorders PSEN2, AD4, STM2, APBB2,FE65L1, NOS3, PLAU, URK, ACE, DCP1, ACE1, MPO, PACIP1, PAXIP1L, PTIP,A2M, BLMH, BMH, PSEN1, AD3); Autism (Mecp2, BZRAP1, MDGA2, Sema5A,Neurexin 1, GLO1, MECP2, RTT, PPMX, MRX16, MRX79, NLGN3, NLGN4,KIAA1260, AUTSX2); Fragile X Syndrome (FMR2, FXR1, FXR2, mGLUR5);Huntington's disease and disease like disorders (HD, IT15, PRNP, PRIP,JPH3, JP3, HDL2, TBP, SCA17); Parkinson disease (NR4A2, NURR1, NOT,TINUR, SNCAIP, TBP, SCA17, SNCA, NACP, PARK1, PARK4, DJ1, PARK7, LRRK2,PARK8, PINK1, PARK6, UCHL1, PARK5, SNCA, NACP, PARK1, PARK4, PRKN,PARK2, PDJ, DBH, NDUFV2); Rett syndrome (MECP2, RTT, PPMX, MRX16, MRX79,CDKL5, STK9, MECP2, RTT, PPMX, MRX16, MRX79, x-Synuclein, DJ-1);Schizophrenia (Neuregulin1 (Nrg1), Erb4 (receptor for Neuregulin),Complexin1 (Cplx1), Tph1 Tryptophan hydroxylase, Tph2, Tryptophanhydroxylase 2, Neurexin 1, GSK3, GSK3a, GSK3b, 5-HTT (Slc6a4), COMT, DRD(Drd1a), SLC6A3, DAOA, DTNBP1, Dao (Dao1)); Secretase Related Disorders(APH-1 (alpha and beta), Presenilin (Psen1), nicastrin, (Ncstn), PEN-2,Nos1, Parp1, Nat1, Nat2); Trinucleotide Repeat Disorders (HTT(Huntington's Dx), SBMA/SMAX1/AR (Kennedy's Dx), FXN/X25 (Friedrich'sAtaxia), ATX3 (Machado- Joseph's Dx), ATXN1 and ATXN2 (spinocerebellarataxias), DMPK (myotonic dystrophy), Atrophin-1 and Atn1 (DRPLA Dx), CBP(Creb-BP - global instability), VLDLR (Alzheimer's), Atxn7, Atxn10).Occular diseases Age-related macular degeneration (Abcr, Ccl2, Cc2, cp(ceruloplasmin), and disorders Timp3, cathepsinD, Vldlr, Ccr2); Cataract(CRYAA, CRYA1, CRYBB2, CRYB2, PITX3, BFSP2, CP49, CP47, CRYAA, CRYA1,PAX6, AN2, MGDA, CRYBA1, CRYB1, CRYGC, CRYG3, CCL, LIM2, MP19, CRYGD,CRYG4, BFSP2, CP49, CP47, HSF4, CTM, HSF4, CTM, MIP, AQP0, CRYAB, CRYA2,CTPP2, CRYBB1, CRYGD, CRYG4, CRYBB2, CRYB2, CRYGC, CRYG3, CCL, CRYAA,CRYA1, GJA8, CX50, CAE1, GJA3, CX46, CZP3, CAE3, CCM1, CAM, KRIT1);Corneal clouding and dystrophy (APOA1, TGFBI, CSD2, CDGG1, CSD, BIGH3,CDG2, TACSTD2, TROP2, M1S1, VSX1, RINX, PPCD, PPD, KTCN, COL8A2, FECD,PPCD2, PIP5K3, CFD); Cornea plana congenital (KERA, CNA2); Glaucoma(MYOC, TIGR, GLC1A, JOAG, GPOA, OPTN, GLC1E, FIP2, HYPL, NRP, CYP1B1,GLC3A, OPA1, NTG, NPG, CYP1B1, GLC3A); Leber congenital amaurosis (CRB1,RP12, CRX, CORD2, CRD, RPGRIP1, LCA6, CORD9, RPE65, RP20, AIPL1, LCA4,GUCY2D, GUC2D, LCA1, CORD6, RDH12, LCA3); Macular dystrophy (ELOVL4,ADMD, STGD2, STGD3, RDS, RP7, PRPH2, PRPH, AVMD, AOFMD, VMD2).

TABLE C CELLULAR FUNCTION GENES PI3K/AKT Signaling PRKCE; ITGAM; ITGA5;IRAK1; PRKAA2; EIF2AK2; PTEN; EIF4E; PRKCZ; GRK6; MAPK1; TSC1; PLK1;AKT2; IKBKB; PIK3CA; CDK8; CDKN1B; NFKB2; BCL2; PIK3CB; PPP2R1A; MAPK8;BCL2L1; MAPK3; TSC2; ITGA1; KRAS; EIF4EBP1; RELA; PRKCD; NOS3; PRKAA1;MAPK9; CDK2; PPP2CA; PIM1; ITGB7; YWHAZ; ILK; TP53; RAF1; IKBKG; RELB;DYRK1A; CDKN1A; ITGB1; MAP2K2; JAK1; AKT1; JAK2; PIK3R1; CHUK; PDPK1;PPP2R5C; CTNNB1; MAP2K1; NFKB1; PAK3; ITGB3; CCND1; GSK3A; FRAP1; SFN;ITGA2; TTK; CSNK1A1; BRAF; GSK3B; AKT3; FOXO1; SGK; HSP90AA1; RPS6KB1ERK/MAPK Signaling PRKCE; ITGAM; ITGA5; HSPB1; IRAK1; PRKAA2; EIF2AK2;RAC1; RAP1A; TLN1; EIF4E; ELK1; GRK6; MAPK1; RAC2; PLK1; AKT2; PIK3CA;CDK8; CREB1; PRKCI; PTK2; FOS; RPS6KA4; PIK3CB; PPP2R1A; PIK3C3; MAPK8;MAPK3; ITGA1; ETS1; KRAS; MYCN; EIF4EBP1; PPARG; PRKCD; PRKAA1; MAPK9;SRC; CDK2; PPP2CA; PIM1; PIK3C2A; ITGB7; YWHAZ; PPP1CC; KSR1; PXN; RAF1;FYN; DYRK1A; ITGB1; MAP2K2; PAK4; PIK3R1; STAT3; PPP2R5C; MAP2K1; PAK3;ITGB3; ESR1; ITGA2; MYC; TTK; CSNK1A1; CRKL; BRAF; ATF4; PRKCA; SRF;STAT1; SGK Glucocorticoid Receptor RAC1; TAF4B; EP300; SMAD2; TRAF6;PCAF; ELK1; Signaling MAPK1; SMAD3; AKT2; IKBKB; NCOR2; UBE2I; PIK3CA;CREB1; FOS; HSPA5; NFKB2; BCL2; MAP3K14; STAT5B; PIK3CB; PIK3C3; MAPK8;BCL2L1; MAPK3; TSC22D3; MAPK10; NRIP1; KRAS; MAPK13; RELA; STAT5A;MAPK9; NOS2A; PBX1; NR3C1; PIK3C2A; CDKN1C; TRAF2; SERPINE1; NCOA3;MAPK14; TNF; RAF1; IKBKG; MAP3K7; CREBBP; CDKN1A; MAP2K2; JAK1; IL8;NCOA2; AKT1; JAK2; PIK3R1; CHUK; STAT3; MAP2K1; NFKB1; TGFBR1; ESR1;SMAD4; CEBPB; JUN; AR; AKT3; CCL2; MMP1; STAT1; IL6; HSP90AA1 AxonalGuidance PRKCE; ITGAM; ROCK1; ITGA5; CXCR4; ADAM12; Signaling IGF1;RAC1; RAP1A; E1F4E; PRKCZ; NRP1; NTRK2; ARHGEF7; SMO; ROCK2; MAPK1; PGF;RAC2; PTPN11; GNAS; AKT2; PIK3CA; ERBB2; PRKCI; PTK2; CFL1; GNAQ;PIK3CB; CXCL12; PIK3C3; WNT11; PRKD1; GNB2L1; ABL1; MAPK3; ITGA1; KRAS;RHOA; PRKCD; PIK3C2A; ITGB7; GLI2; PXN; VASP; RAF1; FYN; ITGB1; MAP2K2;PAK4; ADAM17; AKT1; PIK3R1; GLI1; WNT5A; ADAM10; MAP2K1; PAK3; ITGB3;CDC42; VEGFA; ITGA2; EPHA8; CRKL; RND1; GSK3B; AKT3; PRKCA EphrinReceptor PRKCE; ITGAM; ROCK1; ITGA5; CXCR4; IRAK1; Signaling PRKAA2;EIF2AK2; RAC1; RAP1A; GRK6; ROCK2; MAPK1; PGF; RAC2; PTPN11; GNAS; PLK1;AKT2; DOK1; CDK8; CREB1; PTK2; CFL1; GNAQ; MAP3K14; CXCL12; MAPK8;GNB2L1; ABL1; MAPK3; ITGA1; KRAS; RHOA; PRKCD; PRKAA1; MAPK9; SRC; CDK2;PIM1; ITGB7; PXN; RAF1; FYN; DYRK1A; ITGB1; MAP2K2; PAK4, AKT1; JAK2;STAT3; ADAM10; MAP2K1; PAK3; ITGB3; CDC42; VEGFA; ITGA2; EPHA8; TTK;CSNK1A1; CRKL; BRAF; PTPN13; ATF4; AKT3; SGK Actin Cytoskeleton ACTN4;PRKCE; ITGAM; ROCK1; ITGA5; IRAK1; Signaling PRKAA2; EIF2AK2; RAC1; INS;ARHGEF7; GRK6; ROCK2; MAPK1; RAC2; PLK1; AKT2; PIK3CA; CDK8; PTK2; CFL1;PIK3CB; MYH9; DIAPH1; PIK3C3; MAPK8; F2R; MAPK3; SLC9A1; ITGA1; KRAS;RHOA; PRKCD; PRKAA1; MAPK9; CDK2; PIM1; PIK3C2A; ITGB7; PPP1CC; PXN;VIL2; RAF1; GSN; DYRK1A; ITGB1; MAP2K2; PAK4; PIP5K1A; PIK3R1; MAP2K1;PAK3; ITGB3; CDC42; APC; ITGA2; TTK; CSNK1A1; CRKL; BRAF; VAV3; SGKHuntington's Disease PRKCE; IGF1; EP300; RCOR1; PRKCZ; HDAC4; TGM2;Signaling MAPK1; CAPNS1; AKT2; EGFR; NCOR2; SP1; CAPN2; PIK3CA; HDAC5;CREB1; PRKC1; HSPA5; REST; GNAQ; PIK3CB; PIK3C3; MAPK8; IGF1R; PRKD1;GNB2L1; BCL2L1; CAPN1; MAPK3; CASP8; HDAC2; HDAC7A; PRKCD; HDAC11;MAPK9; HDAC9; PIK3C2A; HDAC3; TP53; CASP9; CREBBP; AKT1; PIK3R1; PDPK1;CASP1; APAF1; FRAP1; CASP2; JUN; BAX; ATF4; AKT3; PRKCA; CLTC; SGK;HDAC6; CASP3 Apoptosis Signaling PRKCE; ROCK1; BID; IRAK1; PRKAA2;EIF2AK2; BAK1; BIRC4; GRK6; MAPK1; CAPNS1; PLK1; AKT2; IKBKB; CAPN2;CDK8; FAS; NFKB2; BCL2; MAP3K14; MAPK8; BCL2L1; CAPN1; MAPK3; CASP8;KRAS; RELA; PRKCD; PRKAA1; MAPK9; CDK2; PIM1; TP53; TNF; RAF1; IKBKG;RELB; CASP9; DYRK1A; MAP2K2; CHUK; APAF1; MAP2K1; NFKB1; PAK3; LMNA;CASP2; BIRC2; TTK; CSNK1A1; BRAF; BAX; PRKCA; SGK; CASP3; BIRC3; PARP1 BCell Receptor RAC1; PTEN; LYN; ELK1; MAPK1; RAC2; PTPN11; SignalingAKT2; IKBKB; PIK3CA; CREB1; SYK; NFKB2; CAMK2A; MAP3K14; PIK3CB; PIK3C3;MAPK8; BCL2L1; ABL1; MAPK3; ETS1; KRAS; MAPK13; RELA; PTPN6; MAPK9;EGR1; PIK3C2A; BTK; MAPK14; RAF1; IKBKG; RELB; MAP3K7; MAP2K2; AKT1;PIK3R1; CHUK; MAP2K1; NFKB1; CDC42; GSK3A; FRAP1; BCL6; BCL10; JUN;GSK3B; ATF4; AKT3; VAV3; RPS6KB1 Leukocyte Extravasation ACTN4; CD44;PRKCE; ITGAM; ROCK1; CXCR4; CYBA; Signaling RAC1; RAP1A; PRKCZ; ROCK2;RAC2; PTPN11; MMP14; PIK3CA; PRKCI; PTK2; PIK3CB; CXCL12; PIK3C3; MAPK8;PRKD1; ABL1; MAPK10; CYBB; MAPK13; RHOA; PRKCD; MAPK9; SRC; PIK3C2A;BTK; MAPK14; NOX1; PXN; VIL2; VASP; ITGB1; MAP2K2; CTNND1; PIK3R1;CTNNB1; CLDN1; CDC42; F11R; ITK; CRKL; VAV3; CTTN; PRKCA; MMP1; MMP9Integrin Signaling ACTN4; ITGAM; ROCK1; ITGA5; RAC1; PTEN; RAP1A; TLN1;ARHGEF7; MAPK1; RAC2; CAPNS1; AKT2; CAPN2; PIK3CA; PTK2; PIK3CB; PIK3C3;MAPK8; CAV1; CAPN1; ABL1; MAPK3; ITGA1; KRAS; RHOA; SRC; PIK3C2A; ITGB7;PPP1CC; ILK; PXN; VASP; RAF1; FYN; ITGB1; MAP2K2; PAK4; AKT1; PIK3R1;TNK2; MAP2K1; PAK3; ITGB3; CDC42; RND3; ITGA2; CRKL; BRAF; GSK3B; AKT3Acute Phase Response IRAK1; SOD2; MYD88; TRAF6; ELK1; MAPK1; PTPN11;Signaling AKT2; IKBKB; PIK3CA; FOS; NFKB2; MAP3K14; PIK3CB; MAPK8;RIPK1; MAPK3; IL6ST; KRAS; MAPK13; IL6R; RELA; SOCS1; MAPK9; FTL; NR3C1;TRAF2; SERPINE1; MAPK14; TNF; RAF1; PDK1; IKBKG; RELB; MAP3K7; MAP2K2;AKT1; JAK2; PIK3R1; CHUK; STAT3; MAP2K1; NFKB1; FRAP1; CEBPB; JUN; AKT3;IL1R1; IL6 PTEN Signaling ITGAM; ITGA5; RAC1; PTEN; PRKCZ; BCL2L11;MAPK1; RAC2; AKT2; EGFR; IKBKB; CBL; PIK3CA; CDKN1B; PTK2; NFKB2; BCL2;PIK3CB; BCL2L1; MAPK3; ITGA1; KRAS; ITGB7; ILK; PDGFRB; INSR; RAF1;IKBKG; CASP9; CDKN1A; ITGB1; MAP2K2; AKT1; PIK3R1; CHUK; PDGFRA; PDPK1;MAP2K1; NFKB1; ITGB3; CDC42; CCND1; GSK3A; ITGA2; GSK3B; AKT3; FOXO1;CASP3; RPS6KB1 p53 Signaling PTEN; EP300; BBC3; PCAF; FASN; BRCA1;GADD45A; BIRC5; AKT2; PIK3CA; CHEK1; TP53INP1; BCL2; PIK3CB; PIK3C3;MAPK8; THBS1; ATR; BCL2L1; E2F1; PMAIP1; CHEK2; TNFRSF10B; TP73; RB1;HDAC9; CDK2; PIK3C2A; MAPK14; TP53; LRDD; CDKN1A; HIPK2; AKT1; PIK3R1;RRM2B; APAF1; CTNNB1; SIRT1; CCND1; PRKDC; ATM; SFN; CDKN2A; JUN; SNAI2;GSK3B; BAX; AKT3 Aryl Hydrocarbon HSPB1; EP300; FASN; TGM2; RXRA; MAPK1;NQO1; Receptor NCOR2; SP1; ARNT; CDKN1B; FOS; CHEK1; Signaling SMARCA4;NFKB2; MAPK8; ALDH1A1; ATR; E2F1; MAPK3; NRIP1; CHEK2; RELA; TP73;GSTP1; RB1; SRC; CDK2; AHR; NFE2L2; NCOA3; TP53; TNF; CDKN1A; NCOA2;APAF1; NFKB1; CCND1; ATM; ESR1; CDKN2A; MYC; JUN; ESR2; BAX; IL6;CYP1B1; HSP90AA1 Xenobiotic Metabolism PRKCE; EP300; PRKCZ; RXRA; MAPK1;NQO1; Signaling NCOR2; PIK3CA; ARNT; PRKCI; NFKB2; CAMK2A; PIK3CB;PPP2R1A; PIK3C3; MAPK8; PRKD1; ALDH1A1; MAPK3; NRIP1; KRAS; MAPK13;PRKCD; GSTP1; MAPK9; NOS2A; ABCB1; AHR; PPP2CA; FTL; NFE2L2; PIK3C2A;PPARGC1A; MAPK14; TNF; RAF1; CREBBP; MAP2K2; PIK3R1; PPP2R5C; MAP2K1;NFKB1; KEAP1; PRKCA; EIF2AK3; IL6; CYP1B1; HSP90AA1 SAPK/JNK SignalingPRKCE; IRAK1; PRKAA2; EIF2AK2; RAC1; ELK1; GRK6; MAPK1; GADD45A; RAC2;PLK1; AKT2; PIK3CA; FADD; CDK8; PIK3CB; PIK3C3; MAPK8; RIPK1; GNB2L1;IRS1; MAPK3; MAPK10; DAXX; KRAS; PRKCD; PRKAA1; MAPK9; CDK2; PIM1;PIK3C2A; TRAF2; TP53; LCK; MAP3K7; DYRK1A; MAP2K2; PIK3R1; MAP2K1; PAK3;CDC42; JUN; TTK; CSNK1A1; CRKL; BRAF; SGK PPAr/RXR Signaling PRKAA2;EP300; INS; SMAD2; TRAF6; PPARA; FASN; RXRA; MAPK1; SMAD3; GNAS; IKBKB;NCOR2; ABCA1; GNAQ; NFKB2; MAP3K14; STAT5B; MAPK8; IRS1; MAPK3; KRAS;RELA; PRKAA1; PPARGC1A; NCOA3; MAPK14; INSR; RAF1; IKBKG; RELB; MAP3K7;CREBBP; MAP2K2; JAK2; CHUK; MAP2K1; NFKB1; TGFBR1; SMAD4; JUN; IL1R1;PRKCA; IL6; HSP90AA1; ADIPOQ NF-KB Signaling IRAK1; EIF2AK2; EP300; INS;MYD88; PRKCZ: TRAF6; TBK1; AKT2; EGFR; IKBKB; PIK3CA; BTRC; NFKB2;MAP3K14; PIK3CB; PIK3C3; MAPK8; RIPK1; HDAC2; KRAS; RELA; PIK3C2A;TRAF2; TLR4: PDGFRB; TNF; INSR; LCK; IKBKG; RELB; MAP3K7; CREBBP; AKT1;PIK3R1; CHUK; PDGFRA; NFKB1; TLR2; BCL10; GSK3B; AKT3; TNFAIP3; IL1R1Neuregulin Signaling ERBB4; PRKCE; ITGAM; ITGA5: PTEN; PRKCZ; ELK1;MAPK1; PTPN11; AKT2; EGFR; ERBB2; PRKCI; CDKN1B; STAT5B; PRKD1; MAPK3;ITGA1; KRAS; PRKCD; STAT5A; SRC; ITGB7; RAF1; ITGB1; MAP2K2; ADAM17;AKT1; PIK3R1; PDPK1; MAP2K1; ITGB3; EREG; FRAP1; PSEN1; ITGA2; MYC;NRG1; CRKL; AKT3; PRKCA; HSP90AA1; RPS6KB1 Wnt & Beta catenin CD44;EP300; LRP6; DVL3; CSNK1E; GJA1; SMO; Signaling AKT2; PIN1; CDH1; BTRC;GNAQ; MARK2; PPP2R1A; WNT11; SRC; DKK1; PPP2CA; SOX6; SFRP2: ILK; LEF1;SOX9; TP53; MAP3K7; CREBBP; TCF7L2; AKT1; PPP2R5C; WNT5A; LRP5; CTNNB1;TGFBR1; CCND1; GSK3A; DVL1; APC; CDKN2A; MYC; CSNK1A1; GSK3B; AKT3; SOX2Insulin Receptor PTEN; INS; EIF4E; PTPN1; PRKCZ; MAPK1; TSC1; SignalingPTPN11; AKT2; CBL; PIK3CA; PRKCI; PIK3CB; PIK3C3; MAPK8; IRS1; MAPK3;TSC2; KRAS; EIF4EBP1; SLC2A4; PIK3C2A; PPP1CC; INSR; RAF1; FYN; MAP2K2;JAK1; AKT1; JAK2; PIK3R1; PDPK1; MAP2K1; GSK3A; FRAP1; CRKL; GSK3B;AKT3; FOXO1; SGK; RPS6KB1 IL-6 Signaling HSPB1; TRAF6; MAPKAPK2; ELK1;MAPK1; PTPN11; IKBKB; FOS; NFKB2: MAP3K14; MAPK8; MAPK3; MAPK10; IL6ST;KRAS; MAPK13; IL6R; RELA; SOCS1; MAPK9; ABCB1; TRAF2; MAPK14; TNF; RAF1;IKBKG; RELB; MAP3K7; MAP2K2; IL8; JAK2; CHUK; STAT3; MAP2K1; NFKB1;CEBPB; JUN; IL1R1; SRF; IL6 Hepatic Cholestasis PRKCE; IRAK1; INS;MYD88; PRKCZ; TRAF6; PPARA; RXRA; IKBKB; PRKCI; NFKB2; MAP3K14; MAPK8;PRKD1; MAPK10; RELA; PRKCD; MAPK9; ABCB1; TRAF2; TLR4; TNF; INSR; IKBKG;RELB; MAP3K7; IL8; CHUK; NR1H2; TJP2; NFKB1; ESR1; SREBF1; FGFR4; JUN;IL1R1; PRKCA; IL6 IGF-1 Signaling IGF1; PRKCZ; ELK1; MAPK1; PTPN11;NEDD4; AKT2; PIK3CA; PRKCI; PTK2; FOS; PIK3CB; PIK3C3; MAPK8; IGF1R;IRS1; MAPK3; IGFBP7; KRAS; PIK3C2A; YWHAZ; PXN; RAF1; CASP9; MAP2K2;AKT1; PIK3R1; PDPK1; MAP2K1; IGFBP2; SFN; JUN; CYR61; AKT3; FOXO1; SRF;CTGF; RPS6KB1 NRF2-mediated PRKCE; EP300; SOD2; PRKCZ; MAPK1; SQSTM1;Oxidative NQO1; PIK3CA; PRKCI; FOS; PIK3CB; PIK3C3; MAPK8; StressResponse PRKD1; MAPK3; KRAS; PRKCD; GSTP1; MAPK9; FTL; NFE2L2; PIK3C2A;MAPK14; RAF1; MAP3K7; CREBBP; MAP2K2; AKT1; PIK3R1; MAP2K1; PPIB; JUN;KEAP1; GSK3B; ATF4; PRKCA; EIF2AK3; HSP90AA1 Hepatic Fibrosis/HepaticEDN1; IGF1; KDR; FLT1; SMAD2; FGFR1; MET; PGF; Stellate Cell ActivationSMAD3; EGFR; FAS; CSF1; NFKB2; BCL2; MYH9; IGF1R; IL6R; RELA; TLR4;PDGFRB; TNF; RELB; IL8; PDGFRA; NFKB1; TGFBR1; SMAD4; VEGFA; BAX; IL1R1;CCL2; HGF; MMP1; STAT1; IL6; CTGF; MMP9 PPAR Signaling EP300; INS;TRAF6; PPARA; RXRA; MAPK1; IKBKB; NCOR2; FOS; NFKB2; MAP3K14; STAT5B;MAPK3; NRIP1; KRAS; PPARG; RELA; STAT5A; TRAF2; PPARGC1A; PDGFRB; TNF;INSR; RAF1; IKBKG; RELB; MAP3K7; CREBBP; MAP2K2; CHUK; PDGFRA; MAP2K1;NFKB1; JUN; IL1R1; HSP90AA1 Fc Epsilon RI Signaling PRKCE; RAC1; PRKCZ;LYN; MAPK1; RAC2; PTPN11; AKT2; PIK3CA; SYK; PRKCI; PIK3CB; PIK3C3;MAPK8; PRKD1; MAPK3; MAPK10; KRAS; MAPK13; PRKCD; MAPK9; PIK3C2A; BTK;MAPK14; TNF; RAF1; FYN; MAP2K2; AKT1; PIK3R1; PDPK1; MAP2K1; AKT3; VAV3;PRKCA G-Protein Coupled PRKCE; RAP1A; RGS16; MAPK1; GNAS; AKT2; IKBKB;Receptor Signaling PIK3CA; CREB1; GNAQ; NFKB2; CAMK2A; PIK3CB; PIK3C3;MAPK3; KRAS; RELA; SRC; PIK3C2A; RAF1; IKBKG; RELB; FYN; MAP2K2; AKT1;PIK3R1; CHUK; PDPK1; STAT3; MAP2K1; NFKB1; BRAF; ATF4; AKT3; PRKCAInositol Phosphate PRKCE; IRAK1; PRKAA2; EIF2AK2; PTEN; GRK6; MetabolismMAPK1; PLK1; AKT2; PIK3CA; CDK8; PIK3CB; PIK3C3; MAPK8; MAPK3; PRKCD;PRKAA1; MAPK9; CDK2; PIM1; PIK3C2A; DYRK1A; MAP2K2; PIP5K1A; PIK3R1;MAP2K1; PAK3; ATM; TTK; CSNK1A1; BRAF; SGK PDGF Signaling EIF2AK2; ELK1;ABL2; MAPK1; PIK3CA; FOS; PIK3CB; PIK3C3; MAPK8; CAV1; ABL1; MAPK3;KRAS; SRC; PIK3C2A; PDGFRB; RAF1; MAP2K2; JAK1; JAK2; PIK3R1; PDGFRA;STAT3; SPHK1; MAP2K1; MYC; JUN; CRKL; PRKCA; SRF; STAT1; SPHK2 VEGFSignaling ACTN4; ROCK1; KDR; FLT1; ROCK2; MAPK1; PGF; AKT2; PIK3CA;ARNT; PTK2; BCL2; PIK3CB; PIK3C3; BCL2L1; MAPK3; KRAS; HIF1A; NOS3;PIK3C2A; PXN; RAF1; MAP2K2; ELAVL1; AKT1; PIK3R1; MAP2K1; SFN; VEGFA;AKT3; FOXO1; PRKCA Natural Killer Cell PRKCE; RAC1; PRKCZ; MAPK1; RAC2;PTPN11; Signaling KIR2DL3; AKT2; PIK3CA; SYK; PRKCI; PIK3CB; PIK3C3;PRKD1; MAPK3; KRAS; PRKCD; PTPN6; PIK3C2A; LCK; RAF1; FYN; MAP2K2; PAK4;AKT1; PIK3R1; MAP2K1; PAK3; AKT3; VAV3; PRKCA Cell Cycle: G1/S HDAC4;SMAD3; SUV39H1; HDAC5; CDKN1B; BTRC; Checkpoint Regulation ATR; ABL1;E2F1; HDAC2; HDAC7A; RB1; HDAC11; HDAC9; CDK2; E2F2; HDAC3; TP53;CDKN1A; CCND1; E2F4; ATM; RBL2; SMAD4; CDKN2A; MYC; NRG1; GSK3B; RBL1;HDAC6 T Cell Receptor RAC1; ELK1; MAPK1; IKBKB; CBL; PIK3CA; FOS;Signaling NFKB2; PIK3CB; PIK3C3; MAPK8; MAPK3; KRAS; RELA, PIK3C2A; BTK;LCK; RAF1; IKBKG; RELB, FYN; MAP2K2; PIK3R1; CHUK; MAP2K1; NFKB1; ITK;BCL10; JUN; VAV3 Death Receptor Signaling CRADD; HSPB1; BID; BIRC4;TBK1; IKBKB; FADD; FAS; NFKB2; BCL2; MAP3K14; MAPK8; RIPK1; CASP8; DAXX;TNFRSF10B; RELA; TRAF2; TNF; IKBKG; RELB; CASP9; CHUK; APAF1; NFKB1;CASP2; BIRC2; CASP3; BIRC3 FGF Signaling RAC1; FGFR1; MET; MAPKAPK2;MAPK1; PTPN11; AKT2; PIK3CA; CREB1; PIK3CB; PIK3C3; MAPK8; MAPK3;MAPK13; PTPN6; PIK3C2A; MAPK14; RAF1; AKT1; PIK3R1; STAT3; MAP2K1;FGFR4; CRKL; ATF4; AKT3; PRKCA; HGF GM-CSF Signaling LYN; ELK1; MAPK1;PTPN11; AKT2; PIK3CA; CAMK2A; STAT5B; PIK3CB; PIK3C3; GNB2L1; BCL2L1;MAPK3; ETS1; KRAS; RUNX1; PIM1; PIK3C2A; RAF1; MAP2K2; AKT1; JAK2;PIK3R1; STAT3; MAP2K1; CCND1; AKT3; STAT1 Amyotrophic Lateral BID; IGF1;RAC1; BIRC4; PGF; CAPNS1; CAPN2; Sclerosis Signaling PIK3CA; BCL2;PIK3CB; PIK3C3; BCL2L1; CAPN1; PIK3C2A; TP53; CASP9; PIK3R1; RAB5A;CASP1; APAF1; VEGFA; BIRC2; BAX; AKT3; CASP3; BIRC3 JAK/Stat SignalingPTPN1; MAPK1; PTPN11; AKT2; PIK3CA; STAT5B; PIK3CB; PIK3C3; MAPK3; KRAS;SOCS1; STAT5A; PTPN6; PIK3C2A; RAF1; CDKN1A; MAP2K2; JAK1; AKT1; JAK2;PIK3R1; STAT3; MAP2K1; FRAP1; AKT3; STAT1 Nicotinate and PRKCE; IRAK1;PRKAA2; EIF2AK2; GRK6; MAPK1; Nicotinamide PLK1; AKT2; CDK8; MAPK8;MAPK3; PRKCD; PRKAA1; Metabolism PBEF1; MAPK9; CDK2; PIM1; DYRK1A;MAP2K2; MAP2K1; PAK3; NT5E; TTK; CSNK1A1; BRAF; SGK Chemokine SignalingCXCR4; ROCK2; MAPK1; PTK2; FOS; CFL1; GNAQ; CAMK2A; CXCL12; MAPK8;MAPK3; KRAS; MAPK13; RHOA; CCR3; SRC; PPP1CC; MAPK14; NOX1; RAF1;MAP2K2; MAP2K1; JUN; CCL2; PRKCA IL-2 Signaling ELK1; MAPK1; PTPN11;AKT2; PIK3CA; SYK; FOS; STAT5B; PIK3CB; PIK3C3; MAPK8; MAPK3; KRAS;SOCS1; STAT5A; PIK3C2A; LCK; RAF1; MAP2K2; JAK1; AKT1; PIK3R1; MAP2K1;JUN; AKT3 Synaptic Long Term PRKCE; IGF1; PRKCZ; PRDX6; LYN; MAPK1;GNAS; Depression PRKCI; GNAQ; PPP2R1A; IGF1R; PRKD1; MAPK3; KRAS; GRN;PRKCD; NOS3; NOS2A; PPP2CA; YWHAZ; RAF1; MAP2K2; PPP2R5C; MAP2K1; PRKCAEstrogen Receptor TAF4B; EP300; CARM1; PCAF; MAPK1; NCOR2; SignalingSMARCA4; MAPK3; NRIP1; KRAS; SRC; NR3C1; HDAC3; PPARGC1A; RBM9; NCOA3;RAF1; CREBBP; MAP2K2; NCOA2; MAP2K1; PRKDC; ESR1; ESR2 ProteinUbiquitination TRAF6; SMURF1; BIRC4; BRCA1; UCHL1; NEDD4; Pathway CBL;UBE2I; BTRC; HSPA5; USP7; USP10; FBXW7; USP9X; STUB1; USP22; B2M; BIRC2;PARK2; USP8; USP1; VHL; HSP90AA1; BIRC3 IL-10 Signaling TRAF6; CCR1;ELK1; IKBKB; SP1; FOS; NFKB2; MAP3K14; MAPK8; MAPK13; RELA; MAPK14; TNF;IKBKG; RELB; MAP3K7; JAK1; CHUK; STAT3; NFKB1; JUN; IL1R1; IL6 VDR/RXRActivation PRKCE; EP300; PRKCZ; RXRA; GADD45A; HES1; NCOR2; SP1; PRKC1;CDKN1B; PRKD1; PRKCD; RUNX2; KLF4; YY1; NCOA3; CDKN1A; NCOA2; SPP1;LRP5; CEBPB; FOXO1; PRKCA TGF-beta Signaling EP300; SMAD2; SMURF1;MAPK1; SMAD3; SMAD1; FOS; MAPK8; MAPK3; KRAS; MAPK9; RUNX2; SERPINE1;RAF1; MAP3K7; CREBBP; MAP2K2; MAP2K1; TGFBR1; SMAD4; JUN; SMAD5Toll-like Receptor IRAK1; EIF2AK2; MYD88; TRAF6; PPARA; ELK1; SignalingIKBKB; FOS; NFKB2; MAP3K14; MAPK8; MAPK13; RELA; TLR4; MAPK14; IKBKG;RELB; MAP3K7; CHUK; NFKB1; TLR2; JUN p38 MAPK Signaling HSPB1; IRAK1;TRAF6; MAPKAPK2; ELK1; FADD; FAS; CREB1; DDIT3; RPS6KA4; DAXX; MAPK13;TRAF2; MAPK14; TNF; MAP3K7; TGFBR1; MYC; ATF4; IL1R1; SRF; STAT1Neurotrophin/TRK NTRK2; MAPK1; PTPN11; PIK3CA; CREB1; FOS; SignalingPIK3CB; PIK3C3; MAPK8; MAPK3; KRAS; PIK3C2A; RAF1; MAP2K2; AKT1; PIK3R1;PDPK1; MAP2K1; CDC42; JUN; ATF4 FXR/RXR Activation INS; PPARA; FASN;RXRA; AKT2; SDC1; MAPK8; APOB; MAPK10; PPARG; MTTP; MAPK9; PPARGC1A;TNF; CREBBP; AKT1; SREBF1; FGFR4; AKT3; FOXO1 Synaptic Long Term PRKCE;RAP1A; EP300; PRKCZ; MAPK1; CREB1; Potentiation PRKCI; GNAQ; CAMK2A;PRKD1; MAPK3; KRAS; PRKCD; PPP1CC; RAF1; CREBBP; MAP2K2; MAP2K1; ATF4;PRKCA Calcium Signaling RAP1A; EP300; HDAC4; MAPK1; HDAC5; CREB1;CAMK2A; MYH9; MAPK3; HDAC2; HDAC7A; HDAC11; HDAC9; HDAC3; CREBBP; CALR;CAMKK2; ATF4; HDAC6 EGF Signaling ELK1; MAPK1; EGFR; PIK3CA; FOS;PIK3CB; PIK3C3; MAPK8; MAPK3; PIK3C2A; RAF1; JAK1; PIK3R1; STAT3;MAP2K1; JUN; PRKCA; SRF; STAT1 Hypoxia Signaling in the EDN1; PTEN;EP300; NQO1; UBE2I; CREB1; ARNT; Cardiovascular System HIF1A; SLC2A4;NOS3; TP53; LDHA; AKT1; ATM; VEGFA; JUN; ATF4; VHL; HSP90AA1 LPS/IL-1Mediated IRAK1; MYD88; TRAF6; PPARA; RXRA; ABCA1, Inhibition MAPK8;ALDH1A1; GSTP1; MAPK9; ABCB1; TRAF2; of RXR Function TLR4; TNF; MAP3K7;NR1H2; SREBF1; JUN; IL1R1 LXR/RXR Activation FASN; RXRA; NCOR2; ABCA1;NFKB2; IRF3; RELA; NOS2A; TLR4; TNF; RELB; LDLR; NR1H2; NFKB1; SREBF1;IL1R1; CCL2; IL6; MMP9 Amyloid Processing PRKCE; CSNK1E; MAPK1; CAPNS1;AKT2; CAPN2; CAPN1; MAPK3; MAPK13; MAPT; MAPK14; AKT1; PSEN1; CSNK1A1;GSK3B; AKT3; APP IL-4 Signaling AKT2; PIK3CA; PIK3CB; PIK3C3; IRS1;KRAS; SOCS1; PTPN6; NR3C1; PIK3C2A; JAK1; AKT1; JAK2; PIK3R1; FRAP1;AKT3; RPS6KB1 Cell Cycle: G2/M DNA EP300; PCAF; BRCA1; GADD45A; PLK1;BTRC; Damage Checkpoint CHEK1; ATR; CHEK2; YWHAZ; TP53; CDKN1A;Regulation PRKDC; ATM; SFN; CDKN2A Nitric Oxide Signaling in KDR; FLT1;PGF; AKT2; PIK3CA; PIK3CB; PIK3C3; the Cardiovascular System CAV1;PRKCD; NOS3; PIK3C2A; AKT1; PIK3R1; VEGFA; AKT3; HSP90AA1 PurineMetabolism NME2; SMARCA4; MYH9; RRM2; ADAR; EIF2AK4; PKM2; ENTPD1;RAD51; RRM2B; TJP2; RAD51C; NT5E; POLD1; NME1 cAMP-mediated RAP1A;MAPK1; GNAS; CREB1; CAMK2A; MAPK3; Signaling SRC; RAF1; MAP2K2; STAT3;MAP2K1; BRAF; ATF4 Mitochondrial SOD2; MAPK8; CASP8; MAPK10; MAPK9;CASP9; Dysfunction PARK7; PSEN1; PARK2; APP; CASP3 Notch Signaling HES1;JAG1; NUMB; NOTCH4; ADAM17; NOTCH2; PSEN1; NOTCH3; NOTCH1; DLL4Endoplasmic Reticulum HSPA5; MAPK8; XBP1; TRAF2; ATF6; CASP9; ATF4;Stress Pathway EIF2AK3; CASP3 Pyrimidine Metabolism NME2; AICDA; RRM2;EIF2AK4; ENTPD1; RRM2B; NT5E; POLD1; NME1 Parkinson's Signaling UCHL1;MAPK8; MAPK13; MAPK14; CASP9; PARK7; PARK2; CASP3 Cardiac & Beta GNAS;GNAQ; PPP2R1A; GNB2L1; PPP2CA; PPP1CC; Adrenergic Signaling PPP2R5CGlycolysis/ HK2; GCK; GPI; ALDH1A1; PKM2; LDHA; HK1 GluconeogenesisInterferon Signaling IRF1; SOCS1; JAK1; JAK2; IFITM1; STAT1; IFIT3 SonicHedgehog ARRB2; SMO; GLI2; DYRK1A; GLI1; GSK3B; DYRKIB SignalingGlycerophospholipid PLD1; GRN; GPAM; YWHAZ; SPHK1; SPHK2 MetabolismPhospholipid PRDX6; PLD1; GRN; YWHAZ; SPHK1; SPHK2 DegradationTryptophan Metabolism SIAH2; PRMT5; NEDD4; ALDH1A1; CYP1B1; SIAH1 LysineDegradation SUV39H1; EHMT2; NSD1; SETD7; PPP2R5C Nucleotide ExcisionERCC5; ERCC4; XPA; XPC; ERCC1 Repair Pathway Starch and Sucrose UCHL1;HK2; GCK; GPI; HK1 Metabolism Aminosugars Metabolism NQO1; HK2; GCK; HK1Arachidonic Acid PRDX6; GRN; YWHAZ; CYP1B1 Metabolism Circadian RhythmCSNK1E; CREB1; ATF4; NR1D1 Signaling Coagulation System BDKRB1; F2R;SERPINE1; F3 Dopamine Receptor PPP2R1A; PPP2CA; PPP1CC; PPP2R5CSignaling Glutathione Metabolism IDH2; GSTP1; ANPEP; IDH1 GlycerolipidMetabolism ALDH1A1; GPAM; SPHK1; SPHK2 Linoleic Acid Metabolism PRDX6;GRN; YWHAZ; CYP1B1 Methionine Metabolism DNMT1; DNMT3B; AHCY; DNMT3APyruvate Metabolism GLO1; ALDH1A1; PKM2; LDHA Arginine and ProlineALDH1A1; NOS3; NOS2A Metabolism Eicosanoid Signaling PRDX6; GRN; YWHAZFructose and Mannose HK2; GCK; HK1 Metabolism Galactose Metabolism HK2;GCK; HK1 Stilbene, Coumarine and PRDX6; PRDX1; TYR Lignin BiosynthesisAntigen Presentation CALR; B2M Pathway Biosynthesis of Steroids NQO1;DHCR7 Butanoate Metabolism ALDH1A1; NLGN1 Citrate Cycle IDH2; IDH1 FattyAcid Metabolism ALDH1A1; CYP1B1 Glycerophospholipid PRDX6; CHKAMetabolism Histidine Metabolism PRMT5; ALDH1A1 Inositol MetabolismERO1L; APEX1 Metabolism of GSTP1; CYP1B1 Xenobiotics by Cytochrome p450Methane Metabolism PRDX6; PRDX1 Phenylalanine PRDX6; PRDX1 MetabolismPropanoate Metabolism ALDH1A1; LDHA Selenoamino Acid PRMT5; AHCYMetabolism Sphingolipid Metabolism SPHK1; SPHK2 Aminophosphonate PRMT5Metabolism Androgen and Estrogen PRMT5 Metabolism Ascorbate and AldarateALDH1A1 Metabolism Bile Acid Biosynthesis ALDH1A1 Cysteine MetabolismLDHA Fatty Acid Biosynthesis FASN Glutamate Receptor GNB2L1 SignalingNRF2-mediated PRDX1 Oxidative Stress Response Pentose Phosphate GPIPathway Pentose and Glucuronate UCHL1 Interconversions RetinolMetabolism ALDH1A1 Riboflavin Metabolism TYR Tyrosine Metabolism PRMT5,TYR Ubiquinone Biosynthesis PRMT5 Valine, Leucine and ALDH1A1 IsoleucineDegradation Glycine, Serine and CHKA Threonine Metabolism LysineDegradation ALDH1A1 Pain/Taste TRPM5; TRPA1 Pain TRPM7; TRPC5; TRPC6;TRPC1; Cnr1; cnr2; Grk2; Trpa1; Pomc; Cgrp; Crf; Pka; Era; Nr2b; TRPM5;Prkaca; Prkacb; Prkar1a; Prkar2a Mitochondrial Function AIF; CytC; SMAC(Diablo); Aifm-1; Aifm-2 Developmental BMP-4; Chordin (Chrd); Noggin(Nog); WNT (Wnt2; Neurology Wnt2b; Wnt3a; Wnt4; Wnt5a; Wnt6; Wnt7b;Wnt8b; Wnt9a; Wnt9b; Wnt10a; Wnt10b; Wnt16); beta-catenin; Dkk-1;Frizzled related proteins; Otx-2; Gbx2; FGF-8; Reelin; Dab1; unc-86(Pou4fl or Brn3a); Numb; Reln

Embodiments of the invention also relate to methods and compositionsrelated to knocking out genes, amplifying genes and repairing particularmutations associated with DNA repeat instability and neurologicaldisorders (Robert D. Wells, Tetsuo Ashizawa, Genetic Instabilities andNeurological Diseases, Second Edition, Academic Press, Oct. 13,2011—Medical). Specific aspects of tandem repeat sequences have beenfound to be responsible for more than twenty human diseases (Newinsights into repeat instability: role of RNA-DNA hybrids. McIvor E I,Polak U, Napierala M. RNA Biol. 2010 September-October; 7(5):551-8). TheCRISPR-Cas system may be harnessed to correct these defects of genomicinstability.

A further aspect of the invention relates to utilizing the CRISPR-Cassystem for correcting defects in the EMP2A and EMP2B genes that havebeen identified to be associated with Lafora disease. Lafora disease isan autosomal recessive condition which is characterized by progressivemyoclonus epilepsy which may start as epileptic seizures in adolescence.A few cases of the disease may be caused by mutations in genes yet to beidentified. The disease causes seizures, muscle spasms, difficultywalking, dementia, and eventually death. There is currently no therapythat has proven effective against disease progression. Other geneticabnormalities associated with epilepsy may also be targeted by theCRISPR-Cas system and the underlying genetics is further described inGenetics of Epilepsy and Genetic Epilepsies, edited by GiulianoAvanzini, Jeffrey L. Noebels, Mariani Foundation PaediatricNeurology:20; 2009).

In yet another aspect of the invention, the CRISPR-Cas system may beused to correct ocular defects that arise from several genetic mutationsfurther described in Genetic Diseases of the Eye, Second Edition, editedby Elias I. Traboulsi, Oxford University Press, 2012.

Several further aspects of the invention relate to correcting defectsassociated with a wide range of genetic diseases which are furtherdescribed on the website of the National Institutes of Health under thetopic subsection Genetic Disorders (website athealth.nih.gov/topic/GeneticDisorders). The genetic brain diseases mayinclude but are not limited to Adrenoleukodystrophy, Agenesis of theCorpus Callosum, Aicardi Syndrome, Alpers' Disease, Alzheimer's Disease,Barth Syndrome, Batten Disease, CADASIL, Cerebellar Degeneration,Fabry's Disease, Gerstmann-Straussler-Scheinker Disease, Huntington'sDisease and other Triplet Repeat Disorders, Leigh's Disease, Lesch-NyhanSyndrome, Menkes Disease, Mitochondrial Myopathies and NINDSColpocephaly. These diseases are further described on the website of theNational Institutes of Health under the subsection Genetic BrainDisorders.

In some embodiments, the condition may be neoplasia. In someembodiments, where the condition is neoplasia, the genes to be targetedare any of those listed in Table A (in this case PTEN and so forth). Insome embodiments, the condition may be Age-related Macular Degeneration.In some embodiments, the condition may be a Schizophrenic Disorder. Insome embodiments, the condition may be a Trinucleotide Repeat Disorder.In some embodiments, the condition may be Fragile X Syndrome. In someembodiments, the condition may be a Secretase Related Disorder. In someembodiments, the condition may be a Prion—related disorder. In someembodiments, the condition may be ALS. In some embodiments, thecondition may be a drug addiction. In some embodiments, the conditionmay be Autism. In some embodiments, the condition may be Alzheimer'sDisease. In some embodiments, the condition may be inflammation. In someembodiments, the condition may be Parkinson's Disease.

Examples of proteins associated with Parkinson's disease include but arenot limited to α-synuclein, DJ-1, LRRK2, PINK1, Parkin, UCHL1,Synphilin-1, and NURR1.

Examples of addiction-related proteins may include ABAT for example.

Examples of inflammation-related proteins may include the monocytechemoattractant protein-1 (MCP1) encoded by the Ccr2 gene, the C-Cchemokine receptor type 5 (CCR5) encoded by the Ccr5 gene, the IgGreceptor IIB (FCGR2b, also termed CD32) encoded by the Fcgr2b gene, orthe Fc epsilon Rlg (FCER1g) protein encoded by the Fcerlg gene, forexample.

Examples of cardiovascular diseases associated proteins may include IL1B(interleukin 1, beta), XDH (xanthine dehydrogenase), TP53 (tumor proteinp53), PTGIS (prostaglandin 12 (prostacyclin) synthase), MB (myoglobin),IL4 (interleukin 4), ANGPT1 (angiopoietin 1), ABCG8 (ATP-bindingcassette, sub-family G (WHITE), member 8), or CTSK (cathepsin K), forexample.

Examples of Alzheimer's disease associated proteins may include the verylow density lipoprotein receptor protein (VLDLR) encoded by the VLDLRgene, the ubiquitin-like modifier activating enzyme 1 (UBA1) encoded bythe UBA1 gene, or the NEDD8-activating enzyme E1 catalytic subunitprotein (UBE1C) encoded by the UBA3 gene, for example.

Examples of proteins associated Autism Spectrum Disorder may include thebenzodiazapine receptor (peripheral) associated protein 1 (BZRAP1)encoded by the BZRAP1 gene, the AF4/FMR2 family member 2 protein (AFF2)encoded by the AFF2 gene (also termed MFR2), the fragile X mentalretardation autosomal homolog 1 protein (FXR1) encoded by the FXR1 gene,or the fragile X mental retardation autosomal homolog 2 protein (FXR2)encoded by the FXR2 gene, for example.

Examples of proteins associated Macular Degeneration may include theATP-binding cassette, sub-family A (ABC1) member 4 protein (ABCA4)encoded by the ABCR gene, the apolipoprotein E protein (APOE) encoded bythe APOE gene, or the chemokine (C-C motif) Ligand 2 protein (CCL2)encoded by the CCL2 gene, for example.

Examples of proteins associated Schizophrenia may include NRG1, ErbB4,CPLX1, TPH1, TPH2, NRXN1, GSK3A, BDNF, DISC1, GSK3B, and combinationsthereof.

Examples of proteins involved in tumor suppression may include ATM(ataxia telangiectasia mutated), ATR (ataxia telangiectasia and Rad3related), EGFR (epidermal growth factor receptor), ERBB2 (v-erb-b2erythroblastic leukemia viral oncogene homolog 2), ERBB3 (v-erb-b2erythroblastic leukemia viral oncogene homolog 3), ERBB4 (v-erb-b2erythroblastic leukemia viral oncogene homolog 4), Notch 1, Notch2,Notch 3, or Notch 4, for example.

Examples of proteins associated with a secretase disorder may includePSENEN (presenilin enhancer 2 homolog (C. elegans)), CTSB (cathepsin B),PSEN1 (presenilin 1), APP (amyloid beta (A4) precursor protein), APH1B(anterior pharynx defective 1 homolog B (C. elegans)), PSEN2 (presenilin2 (Alzheimer disease 4)), or BACE1 (beta-site APP-cleaving enzyme 1),for example.

Examples of proteins associated with Amyotrophic Lateral Sclerosis mayinclude SOD1 (superoxide dismutase 1), ALS2 (amyotrophic lateralsclerosis 2), FUS (fused in sarcoma), TARDBP (TAR DNA binding protein),VAGFA (vascular endothelial growth factor A), VAGFB (vascularendothelial growth factor B), and VAGFC (vascular endothelial growthfactor C), and any combination thereof.

Examples of proteins associated with prion diseases may include SOD1(superoxide dismutase 1), ALS2 (amyotrophic lateral sclerosis 2), FUS(fused in sarcoma), TARDBP (TAR DNA binding protein), VAGFA (vascularendothelial growth factor A), VAGFB (vascular endothelial growth factorB), and VAGFC (vascular endothelial growth factor C), and anycombination thereof.

Examples of proteins related to neurodegenerative conditions in priondisorders may include A2M (Alpha-2-Macroglobulin), AATF (Apoptosisantagonizing transcription factor), ACPP (Acid phosphatase prostate),ACTA2 (Actin alpha 2 smooth muscle aorta), ADAM22 (ADAM metallopeptidasedomain), ADORA3 (Adenosine A3 receptor), or ADRA1D (Alpha-1D adrenergicreceptor for Alpha-1D adrenoreceptor), for example.

Examples of proteins associated with Immunodeficiency may include A2M[alpha-2-macroglobulin]; AANAT [arylalkylamine N-acetyltransferase];ABCA1 [ATP-binding cassette, sub-family A (ABC1), member 1]; ABCA2[ATP-binding cassette, sub-family A (ABC1), member 2]; or ABCA3[ATP-binding cassette, sub-family A (ABC1), member 3]; for example.

Examples of proteins associated with Trinucleotide Repeat Disordersinclude AR (androgen receptor), FMR1 (fragile X mental retardation 1),HTT (huntingtin), or DMPK (dystrophia myotonica-protein kinase), FXN(frataxin), ATXN2 (ataxin 2), for example.

Examples of proteins associated with Neurotransmission Disorders includeSST (somatostatin), NOS1 (nitric oxide synthase 1 (neuronal)), ADRA2A(adrenergic, alpha-2A-, receptor), ADRA2C (adrenergic, alpha-2C-,receptor), TACR1 (tachykinin receptor 1), or HTR2c (5-hydroxytryptamine(serotonin) receptor 2C), for example.

Examples of neurodevelopmental-associated sequences include A2BP1[ataxin 2-binding protein 1], AADAT [aminoadipate aminotransferase],AANAT [arylalkylamine N-acetyltransferase], ABAT [4-aminobutyrateaminotransferase], ABCA1 [ATP-binding cassette, sub-family A (ABC1),member 1], or ABCA13 [ATP-binding cassette, sub-family A (ABC1), member13], for example.

Further examples of preferred conditions treatable with the presentsystem include may be selected from: Aicardi-Goutières Syndrome;Alexander Disease; Allan-Herndon-Dudley Syndrome; POLG-RelatedDisorders; Alpha-Mannosidosis (Type II and III); Alström Syndrome;Angelman; Syndrome; Ataxia-Telangiectasia; NeuronalCeroid-Lipofuscinoses; Beta-Thalassemia; Bilateral Optic Atrophy and(Infantile) Optic Atrophy Type 1; Retinoblastoma (bilateral); CanavanDisease; Cerebrooculofacioskeletal Syndrome 1 [COFS1]; CerebrotendinousXanthomatosis; Cornelia de Lange Syndrome; MAPT-Related Disorders;Genetic Prion Diseases; Dravet Syndrome; Early-Onset Familial AlzheimerDisease; Friedreich Ataxia [FRDA]; Fryns Syndrome; Fucosidosis; FukuyamaCongenital Muscular Dystrophy; Galactosialidosis; Gaucher Disease;Organic Acidemias; Hemophagocytic Lymphohistiocytosis;Hutchinson-Gilford Progeria Syndrome; Mucolipidosis II; Infantile FreeSialic Acid Storage Disease; PLA2G6-Associated Neurodegeneration;Jervell and Lange-Nielsen Syndrome; Junctional Epidermolysis Bullosa;Huntington Disease; Krabbe Disease (Infantile); MitochondrialDNA-Associated Leigh Syndrome and NARP; Lesch-Nyhan Syndrome;LIS1-Associated Lissencephaly; Lowe Syndrome; Maple Syrup Urine Disease;MECP2 Duplication Syndrome; ATP7A-Related Copper Transport Disorders;LAMA2-Related Muscular Dystrophy; Arylsulfatase A Deficiency;Mucopolysaccharidosis Types I, II or III; Peroxisome BiogenesisDisorders, Zellweger Syndrome Spectrum; Neurodegeneration with BrainIron Accumulation Disorders; Acid Sphingomyelinase Deficiency;Niemann-Pick Disease Type C; Glycine Encephalopathy; ARX-RelatedDisorders; Urea Cycle Disorders; COL1A1/2-Related OsteogenesisImperfecta; Mitochondrial DNA Deletion Syndromes; PLP1-RelatedDisorders; Perry Syndrome; Phelan-McDermid Syndrome; Glycogen StorageDisease Type II (Pompe Disease) (Infantile); MAPT-Related Disorders;MECP2-Related Disorders; Rhizomelic Chondrodysplasia Punctata Type 1;Roberts Syndrome; Sandhoff Disease; Schindler Disease—Type 1; AdenosineDeaminase Deficiency; Smith-Lemli-Opitz Syndrome; Spinal MuscularAtrophy; Infantile-Onset Spinocerebellar Ataxia; Hexosaminidase ADeficiency; Thanatophoric Dysplasia Type 1; Collagen Type VI-RelatedDisorders; Usher Syndrome Type I; Congenital Muscular Dystrophy;Wolf-Hirschhorn Syndrome; Lysosomal Acid Lipase Deficiency; andXeroderma Pigmentosum.

As will be apparent, it is envisaged that the present system can be usedto target any polynucleotide sequence of interest. Some examples ofconditions or diseases that might be usefully treated using the presentsystem are included in the Tables above and examples of genes currentlyassociated with those conditions are also provided there. However, thegenes exemplified are not exhaustive.

For example, “wild type StCas9” refers to wild type Cas9 from S.thermophilus, the protein sequence of which is given in the SwissProtdatabase under accession number G3ECR1. Similarly, S. pyogenes Cas9 isincluded in SwissProt under accession number Q99ZW2.

The ability to use CRISPR-Cas systems to perform efficient and costeffective gene editing and manipulation will allow the rapid selectionand comparison of single and and multiplexed genetic manipulations totransform such genomes for improved production and enhanced traits. Inthis regard reference is made to US patents and publications: U.S. Pat.No. 6,603,061—Agrobacterium-Mediated Plant Transformation Method; U.S.Pat. No. 7,868,149—Plant Genome Sequences and Uses Thereof and US2009/0100536—Transgenic Plants with Enhanced Agronomic Traits, all thecontents and disclosure of each of which are herein incorporated byreference in their entirety. In the practice of the invention, thecontents and disclosure of Morrell et al “Crop genomics:advances andapplications” Nat Rev Genet. 2011 Dec. 29; 13(2):85-96 are also hereinincorporated by reference in their entirety.

EXAMPLES

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion. The present examples, along with the methodsdescribed herein are presently representative of preferred embodiments,are exemplary, and are not intended as limitations on the scope of theinvention. Changes therein and other uses which are encompassed withinthe spirit of the invention as defined by the scope of the claims willoccur to those skilled in the art.

Example 1 Methodological Improvement to Simplify Cloning and Delivery

Rather than encoding the U6-promoter and guide RNA on a plasmid,Applicants amplified the U6 promoter with a DNA oligo to add on theguide RNA. The resulting PCR product may be transfected into cells todrive expression of the guide RNA.

Example primer pair that allows the generation a PCR product consistingof U6-promoter::guideRNA targeting human Emx1 locus:

Forward Primer: (SEQ ID NO: 86) AAACTCTAGAgagggcctatttcccatgattc ReversePrimer (carrying the guide RNA, which is underlined): (SEQ ID NO: 87)acctctagAAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATGCTGTTTTGTTTCCAAAACAGCATAGCTCTAAAACCCCTAGTCATTGGAGGTGACGGTGTTTCGTCCTTTCCAC aag

Example 2 Methodological Improvement to Improve Activity

Rather than use pol3 promoters, in particular RNA polymerase III (e.g.U6 or H1 promoters), to express guide RNAs in eukaryotic cells,Applicants express the T7 polymerase in eukaryotic cells to driveexpression of guide RNAs using the T7 promoter.

One example of this system may involve introduction of three pieces ofDNA:

1. expression vector for Cas9

2. expression vector for T7 polymerase

3. expression vector containing guideRNA fused to the T7 promoter

Example 3 Methodological Improvement to Reduce Toxicity of Cas9:Delivery of Cas9 in the Form of mRNA

Delivery of Cas9 in the form of mRNA enables transient expression ofCas9 in cells, to reduce toxicity. For example, humanized SpCas9 may beamplified using the following primer pair:

Forward Primer (to add on T7 promoter for in vitro transcription): (SEQID NO: 88) TAATACGACTCACTATAGGAAGTGCGCCACCATGGCCCCAAAGAAGAAGC GG ReversePrimer (to add on polyA tail): (SEQ ID NO: 89)GGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTttcttaCTTTTTCTTTTT TGCCTGGCCG

Applicants transfect the Cas9 mRNA into cells with either guide RNA inthe form of RNA or DNA cassettes to drive guide RNA expression ineukaryotic cells.

Example 4 Methodological Improvement to Reduce Toxicity of Cas9: Use ofan Inducible Promoter

Applicants transiently turn on Cas9 expression only when it is neededfor carrying out genome modification. Examples of inducible systeminclude tetracycline inducible promoters (Tet-On or Tet-Off), smallmolecule two-hybrid transcription activations systems (FKBP, ABA, etc),or light inducible systems (Phytochrome, LOV domains, or cryptochrome).

Example 5 Improvement of the Cas9 System for In Vivo Application

Applicants conducted a Metagenomic search for a Cas9 with smallmolecular weight. Most Cas9 homologs are fairly large. For example theSpCas9 is around 1368aa long, which is too large to be easily packagedinto viral vectors for delivery. A graph representing the lengthdistribution of Cas9 homologs is generated from sequences deposited inGenBank (FIG. 5). Some of the sequences may have been mis-annotated andtherefore the exact frequency for each length may not necessarily beaccurate. Nevertheless it provides a glimpse at distribution of Cas9proteins and suggest that there are shorter Cas9 homologs.

Through computational analysis, Applicants found that in the bacterialstrain Campylobacter, there are two Cas9 proteins with less than 1000amino acids. The sequence for one Cas9 from Campylobacter jejuni ispresented below. At this length, CjCas9 can be easily packaged into AAV,lentiviruses, Adenoviruses, and other viral vectors for robust deliveryinto primary cells and in vivo in animal models. In a preferredembodiment of the invention, the Cas9 protein from S. aureus is used.

>Campylobacter jejuni Cas9 (CjCas9) (SEQ ID NO: 90)MARILAFDIGISSIGWAFSENDELKDCGVRIFTKVENPKTGESLALPRRLARSARKRLARRKARLNHLKHLIANEFKLNYEDYQSFDESLAKAYKGSLISPYELRFRALNELLSKQDFARVILHIAKRRGYDDIKNSDDKEKGAILKAIKQNEEKLANYQSVGEYLYKEYFQKFKENSKEFTNVRNKKESYERCIAQSFLKDELKLIFKKQREFGFSFSKKFEEEVLSVAFYKRALKDFSHLVGNCSFFTDEKRAPKNSPLAFMFVALTRIINLLNNLKNTEGILYTKDDLNALLNEVLKNGTLTYKQTKKLLGLSDDYEFKGEKGTYFIEFKKYKEFIKALGEHNLSQDDLNEIAKDITLIKDEIKLKKALAKYDLNQNQIDSLSKLEFKDHLNISFKALKLVTPLMLEGKKYDEACNELNLKVAINEDKKDFLPAFNETYYKDEVTNPVVLRAIKEYRKVLNALLKKYGKVHKINIELAREVGKNHSQRAKIEKEQNENYKAKKDAELECEKLGLKINSKNILKLRLFKEQKEFCAYSGEKIKISDLQDEKMLEIDHIYPYSRSFDDSYMNKVLVFTKQNQEKLNQTPFEAFGNDSAKWQKIEVLAKNLPTKKQKRILDKNYKDKEQKNFKDRNLNDTRYIARLVLNYTKDYLDFLPLSDDENTKLNDTQKGSKVHVEAKSGMLTSALRHTWGFSAKDRNNHLHHAIDAVIIAYANNSIVKAFSDFKKEQESNSAELYAKKISELDYKNKRKFFEPFSGFRQKVLDKIDEIFVSKPERKKPSGALHEETFRKEEEFYQSYGGKEGVLKALELGKIRKVNGKIVKNGDMFRVDIFKHKKTNKFYAVPIYTMDFALKVLPNKAVARSKKGEIKDWILMDENYEFCFSLYKDSLILIQTKDMQEPEFVYYNAFTSSTVSLIVSKHDNKFETLSKNQKILFKNANEKEVIAKSIGIQNLKVFEKYIVSALGEVTKAEFRQREDFKK. The putative tracrRNA element forthis CjCas9 is: (SEQ ID NO: 91)TATAATCTCATAAGAAATTTAAAAAGGGACTAAAATAAAGAGTTTGCGGGACTCTGCGGGGTTACAATCCCCTAAAACCGCTTTTAAAATT The Direct Repeat sequenceis: (SEQ ID NO: 92) ATTTTACCATAAAGAAATTTAAAAAGGGACTAAAAC An example of achimeric guideRNA for CjCas9 is: (SEQ ID NO: 93)NNNNNNNNNNNNNNNNNNNNGUUUUAGUCCCGAAAGGGACUAAAAUAAAGAGUUUGCGGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU

Example 6 Cas9 Optimization

For enhanced function or to develop new functions, Applicants generatechimeric Cas9 proteins by combining fragments from different Cas9homologs. For example, two example chimeric Cas9 proteins:

For example, Applicants fused the N-term of StlCas9 (fragment from thisprotein is in bold) with C-term of SpCas9 (fragment from this protein isunderlined).

>St1(N)Sp(C)Cas9 (SEQ ID NO: 94)MSDLVLGLDIGIGSVGVGILNKVTGEIIHKNSRIFPAAQAENNLVRRTNRQGRRLARRKKHRRVRLNRLFEESGLITDFTKISINLNPYQLRVKGLTDELSNEELFIALKNMVKHRGISYLDDASDDGNSSVGDYAQIVKENSKQLETKTPGQIQLERYQTYGQLRGDFTVEKDGKKHRLINVFPTSAYRSEALRILQTQQEFNPQITDEFINRYLEILTGKRKYYHGPGNEKSRTDYGRYRTSGETLDNIFGILIGKCTFYPDEFRAAKASYTAQEFNLLNDLNNLTVPTETKKLSKEQKNQIINYVKNEKAMGPAKLFKYIAKLLSCDVADIKGYRIDKSGKAEIHTFEAYRKMKTLETLDIEQMDRETLDKLAYVLTLNTEREGIQEALEHEFADGSFSQKQVDELVQFRKANSSIFGKGWHNFSVKLMMELIPELYETSEEQMTILTRLGKQKTTSSSNKTKYIDEKLLTEEIYNPVVAKSVRQAIKIVNAAIKE YGDFDNIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD >Sp(N)St1(C)Cas9 (SEQ ID NO: 95)MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV MGRHKPENIVIEMARETNEDDEKKAIQKIQKANKDEKDAAMLKAANQYNGKAELPHSVFHGHKQLATKIRLWHQQGERCLYTGKTISIHDLINNSNQFEVDHILPLSITFDDSLANKVLVYATANQEKGQRTPYQALDSMDDAWSFRELKAFVRESKTLSNKKKEYLLTEEDISKFDVRKKFIERNLVDTRYASRVVLNALQEHFRAHKIDTKVSVVRGQFTSQLRRHWGIEKTRDTYHHHAVDALIIAASSQLNLWKKQKNTLVSYSEDQLLDIETGELISDDEYKESVFKAPYQHFVDTLKSKEFEDSILFSYQVDSKFNRKISDATIYATRQAKVGKDKADETYVLGKIKDIYTQDGYDAFMKIYKKDKSKFLMYRHDPQTFEKVIEPILENYPNKQINEKGKEVPCNPFLKYKEEHGYIRKYSKKGNGPEIKSLKYYDSKLGNHIDITPKDSNNKVVLQSVSPWRADVYFNKTTGKYEILGLKYADLQFEKGTGTYKISQEKYNDIKKKEGVDSDSEFKFTLYKNDLLLVKDTETKEQQLFRFLSRTMPKQKHYVELKPYDKQKFEGGEALIKVLGNVANSGQCKKGLGKSNISIYKVRTDVLGNQHIIKNEGDKPKLDF

The benefit of making chimeric Cas9 include:

reduce toxicity

improve expression in eukaryotic cells

enhance specificity

reduce molecular weight of protein, make protein smaller by combiningthe smallest domains from different Cas9 homologs.

Altering the PAM sequence requirement

Example 7 Utilization of Cas9 as a Generic DNA Binding Protein

Applicants used Cas9 as a generic DNA binding protein by mutating thetwo catalytic domains (D10 and H840) responsible for cleaving bothstrands of the DNA target. In order to upregulate gene transcription ata target locus Applicants fused the transcriptional activation domain(VP64) to Cas9. Applicants hypothesized that it would be important tosee strong nuclear localization of the Cas9-VP64 fusion protein becausetranscription factor activation strength is a function of time spent atthe target. Therefore, Applicants cloned a set of Cas9-VP64-GFPconstructs, transfected them into 293 cells and assessed theirlocalization under a fluorescent microscope 12 hours post-transfection.

The same constructs were cloned as a 2A-GFP rather than a direct fusionin order to functionally test the constructs without a bulky GFP presentto interfere. Applicants elected to target the Sox2 locus with the Cas9transactivator because it could be useful for cellular reprogram and thelocus has already been validated as a target for TALE-TF mediatedtranscriptional activation. For the Sox2 locus Applicants chose eighttargets near the transcriptional start site (TSS). Each target was 20 bplong with a neighboring NGG protospacer adjacent motif (PAM). EachCas9-VP64 construct was co-transfected with each PCR generated chimericcrispr RNA (chiRNA) in 293 cells. 72 hours post transfection thetranscriptional activation was assessed using RT-qPCR.

To further optimize the transcriptional activator, Applicants titratedthe ratio of chiRNA (Sox2.1 and Sox2.5) to Cas9(NLS-VP64-NLS-hSpCas9-NLS-VP64-NLS), transfected into 293 cells, andquantified using RT-qPCR. These results indicate that Cas9 can be usedas a generic DNA binding domain to upregulate gene transcription at atarget locus.

Applicants designed a second generation of constructs. (see list below).

-   pLenti-EF1a-GFP-2A-6×His-NLS-VP64-NLS-hSpCsn1(D10A, H840A)-NLS    (“6×His” disclosed as SEQ ID NO: 96)-   pLenti-EF1a-GFP-2A-6×His-NLS-VP64-NLS-hSpCsn1(D10A, H840A) (“6×His”    disclosed as SEQ ID NO: 96)-   pLenti-EF1a-GFP-2A-6×His-NLS-VP64-NLS-NLS-hSpCsn1 (D10A, H840A)    (“6×His” disclosed as SEQ ID NO: 96)-   pLenti-EF1a-GFP-2A-6×His-NLS-hSpCsn1(D10A, H840A)-NLS (“6×His”    disclosed as SEQ ID NO: 96)-   pLenti-EF1a-GFP-2A-6×His-NLS-hSpCsn1(D10A, H840A) (“6×His” disclosed    as SEQ ID NO: 96)-   pLenti-EF1a-GFP-2A-6×His-NLS-NLS-hSpCsn1(D10A, H840A) (“6×His”    disclosed as SEQ ID NO: 96)

Applicants use these constructs to assess transcriptional activation(VP64 fused constructs) and repression (Cas9 only) by RT-qPCR.Applicants assess the cellular localization of each construct usinganti-His antibody, nuclease activity using a Surveyor nuclease assay,and DNA binding affinity using a gel shift assay. In a preferredembodiment of the invention, the gel shift assay is an EMSA gel shiftassay.

Example 8 Cas9 Transgenic and Knock in Mice

To generate a mouse that expresses the Cas9 nuclease Applicants submittwo general strategies, transgenic and knock in. These strategies may beapplied to generate any other model organism of interest, for e.g. Rat.For each of the general strategies Applicants made a constitutivelyactive Cas9 and a Cas9 that is conditionally expressed (Cre recombinasedependent). The constitutively active Cas9 nuclease is expressed in thefollowing context: pCAG-NLS-Cas9-NLS-P2A-EGFP-WPRE-bGHpolyA. pCAG is thepromoter, NLS is a nuclear localization signal, P2A is the peptidecleavage sequence, EGFP is enhanced green fluorescent protein, WPRE isthe woodchuck hepatitis virus posttranscriptional regulatory element,and bGHpolyA is the bovine growth hormone poly-A signal sequence (FIGS.7A-B). The conditional version has one additional stop cassette element,loxP-SV40 polyA x3-loxP, after the promoter and before NLS-Cas9-NLS(i.e. pCAG-loxP-SV4OpolyAx3-loxP-NLS-Cas9-NLS-P2A-EGFP-WPRE-bGHpolyA).The important expression elements can be visualized as in FIG. 8. Theconstitutive construct should be expressed in all cell types throughoutdevelopment, whereas, the conditional construct will only allow Cas9expression when the same cell is expressing the Cre recombinase. Thislatter version will allow for tissue specific expression of Cas9 whenCre is under the expression of a tissue specific promoter. Moreover,Cas9 expression could be induced in adult mice by putting Cre under theexpression of an inducible promoter such as the TET on or off system.

Validation of Cas9 constructs: Each plasmid was functionally validatedin three ways: 1) transient transfection in 293 cells followed byconfirmation of GFP expression; 2) transient transfection in 293 cellsfollowed by immunofluorescence using an antibody recognizing the P2Asequence; and 3) transient transfection followed by Surveyor nucleaseassay. The 293 cells may be 293FT or 293 T cells depending on the cellsthat are of interest. In a preferred embodiment the cells are 293FTcells. The results of the Surveyor were run out on the top and bottomrow of the gel for the conditional and constitutive constructs,respectively. Each was tested in the presence and absence of chimericRNA targeted to the hEMX1 locus (chimeric RNA hEMX1.1). The resultsindicate that the construct can successfully target the hEMX1 locus onlyin the presence of chimeric RNA (and Cre in the conditional case). Thegel was quantified and the results are presented as average cuttingefficiency and standard deviation for three samples.

Transgenic Cas9 mouse: To generate transgenic mice with constructs,Applicants inject pure, linear DNA into the pronucleus of a zygote froma pseudo pregnant CB56 female. Founders are identified, genotyped, andbackcrossed to CB57 mice. The constructs were successfully cloned andverified by Sanger sequencing.

Knock in Cas9 mouse: To generate Cas9 knock in mice Applicants targetthe same constitutive and conditional constructs to the Rosa26 locus.Applicants did this by cloning each into a Rosa26 targeting vector withthe following elements: Rosa26 short homologyarm—constitutive/conditional Cas9 expression cassette—pPGK-Neo-Rosa26long homology arm—pPGK-DTA. pPGK is the promoter for the positiveselection marker Neo, which confers resistance to neomycin, a 1 kb shortarm, a 4.3 kb long arm, and a negative selection diphtheria toxin (DTA)driven by PGK.

The two constructs were electroporated into R1 mESCs and allowed to growfor 2 days before neomycin selection was applied. Individual coloniesthat had survived by days 5-7 were picked and grown in individual wells.5-7 days later the colonies were harvested, half were frozen and theother half were used for genotyping. Genotyping was done by genomic PCR,where one primer annealed within the donor plasmid (AttpF) and the otheroutside of the short homology arm (Rosa26-R) Of the 22 coloniesharvested for the conditional case, 7 were positive (Left). Of the 27colonies harvested for the constitutive case, zero were positive(Right). It is likely that Cas9 causes some level of toxicity in themESC and for this reason there were no positive clones. To test thisApplicants introduced a Cre expression plasmid into correctly targetedconditional Cas9 cells and found very low toxicity after many days inculture. The reduced copy number of Cas9 in correctly targetedconditional Cas9 cells (1-2 copies per cell) is enough to allow stableexpression and relatively no cytotoxicity. Moreover, this data indicatesthat the Cas9 copy number determines toxicity. After electroporationeach cell should get several copies of Cas9 and this is likely why nopositive colonies were found in the case of the constitutive Cas9construct. This provides strong evidence that utilizing a conditional,Cre-dependent strategy should show reduced toxicity. Applicants injectcorrectly targeted cells into a blastocyst and implant into a femalemouse. Chimerics are identified and backcrossed. Founders are identifiedand genotyped.

Utility of the conditional Cas9 mouse: Applicants have shown in 293cells that the Cas9 conditional expression construct can be activated byco-expression with Cre. Applicants also show that the correctly targetedR1 mESCs can have active Cas9 when Cre is expressed. Because Cas9 isfollowed by the P2A peptide cleavage sequence and then EGFP Applicantsidentify successful expression by observing EGFP. This same concept iswhat makes the conditional Cas9 mouse so useful. Applicants may crosstheir conditional Cas9 mouse with a mouse that ubiquitously expressesCre (ACTB-Cre line) and may arrive at a mouse that expresses Cas9 inevery cell. It should only take the delivery of chimeric RNA to inducegenome editing in embryonic or adult mice. Interestingly, if theconditional Cas9 mouse is crossed with a mouse expressing Cre under atissue specific promoter, there should only be Cas9 in the tissues thatalso express Cre. This approach may be used to edit the genome in onlyprecise tissues by delivering chimeric RNA to the same tissue.

Example 9 Cas9 Diversity and Chimeric RNAs

The CRISPR-Cas system is an adaptive immune mechanism against invadingexogenous DNA employed by diverse species across bacteria and archaea.The type II CRISPR-Cas system consists of a set of genes encodingproteins responsible for the “acquisition” of foreign DNA into theCRISPR locus, as well as a set of genes encoding the “execution” of theDNA cleavage mechanism; these include the DNA nuclease (Cas9), anon-coding transactivating cr-RNA (tracrRNA), and an array of foreignDNA-derived spacers flanked by direct repeats (crRNAs). Upon maturationby Cas9, the tracrRNA and crRNA duplex guide the Cas9 nuclease to atarget DNA sequence specified by the spacer guide sequences, andmediates double-stranded breaks in the DNA near a short sequence motifin the target DNA that is required for cleavage and specific to eachCRISPR-Cas system. The type II CRISPR-Cas systems are found throughoutthe bacterial kingdom and highly diverse in in Cas9 protein sequence andsize, tracrRNA and crRNA direct repeat sequence, genome organization ofthese elements, and the motif requirement for target cleavage. Onespecies may have multiple distinct CRISPR-Cas systems.

Applicants evaluated 207 putative Cas9s from bacterial speciesidentified based on sequence homology to known Cas9s and structuresorthologous to known subdomains, including the HNH endonuclease domainand the RuvC endonuclease domains [information from the Eugene Kooninand Kira Makarova]. Phylogenetic analysis based on the protein sequenceconservation of this set revealed five families of Cas9s, includingthree groups of large Cas9s (˜1400 amino acids) and two of small Cas9s(˜1100 amino acids) (FIGS. 3 and 4A-F).

Applicants have also optimized Cas9 guide RNA using in vitro methods.

Example 10 Cas9 Mutations

In this example, Applicants show that the following mutations canconvert SpCas9 into a nicking enzyme: D10A, E762A, H840A, N854A, N863A,D986A.

Applicants provide sequences showing where the mutation points arelocated within the SpCas9 gene (FIG. 6A-M). Applicants also show thatthe nickases are still able to mediate homologous recombination.Furthermore, Applicants show that SpCas9 with these mutations(individually) do not induce double strand break.

Cas9 orthologs all share the general organization of 3-4 RuvC domainsand a HNH domain. The 5′ most RuvC domain cleaves the non-complementarystrand, and the HNH domain cleaves the complementary strand. Allnotations are in reference to the guide sequence.

The catalytic residue in the 5′ RuvC domain is identified throughhomology comparison of the Cas9 of interest with other Cas9 orthologs(from S. pyogenes type II CRISPR locus, S. thermophilus CRISPR locus 1,S. thermophilus CRISPR locus 3, and Franciscilla novicida type II CRISPRlocus), and the conserved Asp residue is mutated to alanine to convertCas9 into a complementary-strand nicking enzyme. Similarly, theconserved His and Asn residues in the HNH domains are mutated to Alanineto convert Cas9 into a non-complementary-strand nicking enzyme.

Example 11 Cas9 Transcriptional Activation and Cas9 Repressor

Cas9 Transcriptional Activation

A second generation of constructs have been designed and are in thepipeline to be tested (Table 1). These constructs will be used to assesstranscriptional activation (VP64 fused constructs) and repression (Cas9only) by RT-qPCR. Applicants will also assess the cellular localizationof each construct using anti-His antibody, nuclease activity using aSurveyor nuclease assay, and DNA binding affinity using a gel shiftassay.

Cas Repressor

It has been shown previously that dCas9 can be used as a generic DNAbinding domain to repress gene expression. Applicants report an improveddCas9 design as well as dCas9 fusions to the repressor domains KRAB andSID4x. From the plasmid library created for modulating transcriptionusing Cas9 in Table 1, the following repressor plasmids werefunctionally characterized by qPCR: pXRP27, pXRP28, pXRP29, pXRP48,pXRP49, pXRP50, pXRP51, pXRP52, pXRP53, pXRP56, pXRP58, pXRP59, pXRP61,and pXRP62.

Each dCas9 repressor plasmid was co-transfected with two guide RNAstargeted to the coding strand of the beta-catenin gene. RNA was isolated72 hours after transfection and gene expression was quantified byRT-qPCR. The endogenous control gene was GAPDH. Two validated shRNAswere used as positive controls. Negative controls were certain plasmidstransfected without gRNA, these are denoted as “pXRP## control”. Theplasmids pXRP28, pXRP29, pXRP48, and pXRP49 could repress thebeta-catenin gene when using the specified targeting strategy. Theseplasmids correspond to dCas9 without a functional domain (pXRP28 andpXRP28) and dCas9 fused to SID4x (pXRP48 and pXRP49).

Further work investigates: repeating the above experiment, targetingdifferent genes, utilizing other gRNAs to determine the optimaltargeting position, and multiplexed repression.

TABLE 1 (Table 1 discloses “GGGGS₃” as SEQ ID NO: 97, “EAAAK₃” as SEQ IDNO: 98 and “GGGGGS₃” as SEQ ID NO: 99)pXRP024-pLenti2-EF1a-VP64-NLS-FLAG-Linker-dCas9-NLS-gLuc-2A-GFP-WPREpXRP025-pLenti2-EF1a-VP64-NLS-GGGGS₃Linker-dCas9-NLS-gLuc-2A-GFP-WPREpXRP026-pLenti2-EF1a-VP64-NLS-EAAAK₃Linker-dCas9-NLS-gLuc-2A-GFP-WPREpXRP027-pLenti2-EF1a-NLS-FLAG-Linker-dCas9-NLS-gLuc-2A-GFP-WPREpXRP028-pLenti2-EF1a-NLS-GGGGS₃Linker-dCas9-NLS-gLuc-2A-GFP-WPREpXRP029-pLenti2-EF1a-NLS-EAAAK₃Linker-dCas9-NLS-gLuc-2A-GFP-WPREpXRP030-pLenti2-pSV40-VP64-NLS-FLAG-Linker-dCas9-NLS-gLuc-2A-GFP-WPREpXRP031-pLenti2-pPGK-VP64-NLS-FLAG-Linker-dCas9-NLS-gLuc-2A-GFP-WPREpXRP032-pLenti2-LTR-VP64-NLS-FLAG-Linker-dCas9-NLS-gLuc-2A-GFP-WPREpXRP033-pLenti2-pSV40-VP64-NLS-GGGGS₃Linker-dCas9-NLS-gLuc-2A-GFP-WPREpXRP034-pLenti2-pPGK-VP64-NLS-GGGGS₃Linker-dCas9-NLS-gLuc-2A-GFP-WPREpXRP035-pLenti2-LTR-VP64-NLS-GGGGS₃Linker-dCas9-NLS-gLuc-2A-GFP-WPREpXRP036-pLenti2-pSV40-VP64-NLS-EAAAK₃Linker-dCas9-NLS-gLuc-2A-GFP-WPREpXRP037-pLenti2-pPGK-VP64-NLS-EAAAK₃Linker-dCas9-NLS-gLuc-2A-GFP-WPREpXRP038-pLenti2-LTR-VP64-NLS-EAAAK₃Linker-dCas9-NLS-gLuc-2A-GFP-WPREpXRP048-pLenti2-EF1a-SID4x-NLS-FLAG-Linker-dCas9-NLS-gLuc-2A-GFP-WPREpXRP049-pLenti2-EF1a-SID4X-NLS-GGGGS₃Linker-dCas9-NLS-gLuc-2A-GFP-WPREpXRP050-pLenti2-EF1a-SID4X-NLS-EAAAK₃Linker-dCas9-NLS-gLuc-2A-GFP-WPREpXRP051-pLenti2-EF1a-KRAB-NLS-FLAG-Linker-dCas9-NLS-gLuc-2A-GFP-WPREpXRP052-pLenti2-EF1a-KRAB-NLS-GGGGS₃Linker-dCas9-NLS-gLuc-2A-GFP-WPREpXRP053-pLenti2-EF1a-KRAB-NLS-EAAAK₃Linker-dCas9-NLS-gLuc-2A-GFP-WPREpXRP054-pLenti2-EF1a-dCas9-Linker-FLAG-NLS-VP64-gLuc-2A-GFP-WPREpXRP055-pLenti2-EF1a-dCas9-Linker-FLAG-NLS-SID4X-gLuc-2A-GFP-WPREpXRP056-pLenti2-EF1a-dCas9-Linker-FLAG-NLS-KRAB-gLuc-2A-GFP-WPREpXRP057-pLenti2-EF1a-dCas9-GGGGGS₃-NLS-VP64-gLuc-2A-GFP-WPREpXRP058-pLenti2-EF1a-dCas9-GGGGGS₃-NLS-SID4X-gLuc-2A-GFP-WPREpXRP059-pLenti2-EF1a-dCas9-GGGGGS₃-NLS-KRAB-gLuc-2A-GFP-WPREpXRP060-pLenti2-EF1a-dCas9-EAAAK₃-NLS-VP64-gLuc-2A-GFP-WPREpXRP061-pLenti2-EF1a-dCas9-EAAAK₃-NLS-SID4X-gLuc-2A-GFP-WPREpXRP062-pLenti2-EF1a-dCas9-EAAAK₃-NLS-KRAB-gLuc-2A-GFP-WPREpXRP024-pLenti2-EF1a-VP64-NLS-FLAG-Linker-Cas9-NLS-gLuc-2A-GFP-WPREpXRP025-pLenti2-EF1a-VP64-NLS-GGGGS₃Linker-Cas9-NLS-gLuc-2A-GFP-WPREpXRP026-pLenti2-EF1a-VP64-NLS-EAAAK₃Linker-Cas9-NLS-gLuc-2A-GFP-WPREpXRP027-pLenti2-EF1a-NLS-FLAG-Linker-Cas9-NLS-gLuc-2A-GFP-WPREpXRP028-pLenti2-EF1a-NLS-GGGGS₃Linker-Cas9-NLS-gLuc-2A-GFP-WPREpXRP029-pLenti2-EF1a-NLS-EAAAK₃Linker-Cas9-NLS-gLuc-2A-GFP-WPREpXRP030-pLenti2-pSV40-VP64-NLS-FLAG-Linker-Cas9-NLS-gLuc-2A-GFP-WPREpXRP031-pLenti2-pPGK-VP64-NLS-FLAG-Linker-Cas9-NLS-gLuc-2A-GFP-WPREpXRP032-pLenti2-LTR-VP64-NLS-FLAG-Linker-Cas9-NLS-gLuc-2A-GFP-WPREpXRP033-pLenti2-pSV40-VP64-NLS-GGGGS₃Linker-Cas9-NLS-gLuc-2A-GFP-WPREpXRP034-pLenti2-pPGK-VP64-NLS-GGGGS₃Linker-Cas9-NLS-gLuc-2A-GFP-WPREpXRP035-pLenti2-LTR-VP64-NLS-GGGGS₃Linker-Cas9-NLS-gLuc-2A-GFP-WPREpXRP036-pLenti2-pSV40-VP64-NLS-EAAAK₃Linker-Cas9-NLS-gLuc-2A-GFP-WPREpXRP037-pLenti2-pPGK-VP64-NLS-EAAAK₃Linker-Cas9-NLS-gLuc-2A-GFP-WPREpXRP038-pLenti2-LTR-VP64-NLS-EAAAK₃Linker-Cas9-NLS-gLuc-2A-GFP-WPREpXRP048-pLenti2-EF1a-SID4x-NLS-FLAG-Linker-Cas9-NLS-gLuc-2A-GFP-WPREpXRP049-pLenti2-EF1a-SID4X-NLS-GGGGS₃Linker-Cas9-NLS-gLuc-2A-GFP-WPREpXRP050-pLenti2-EF1a-SID4X-NLS-EAAAK₃Linker-Cas9-NLS-gLuc-2A-GFP-WPREpXRP051-pLenti2-EF1a-KRAB-NLS-FLAG-Linker-Cas9-NLS-gLuc-2A-GFP-WPREpXRP052-pLenti2-EF1a-KRAB-NLS-GGGGS₃Linker-Cas9-NLS-gLuc-2A-GFP-WPREpXRP053-pLenti2-EF1a-KRAB-NLS-EAAAK₃Linker-Cas9-NLS-gLuc-2A-GFP-WPREpXRP054-pLenti2-EF1a-Cas9-Linker-FLAG-NLS-VP64-gLuc-2A-GFP-WPREpXRP055-pLenti2-EF1a-Cas9-Linker-FLAG-NLS-SID4X-gLuc-2A-GFP-WPREpXRP056-pLenti2-EF1a-Cas9-Linker-FLAG-NLS-KRAB-gLuc-2A-GFP-WPREpXRP057-pLenti2-EF1a-Cas9-GGGGGS₃-NLS-VP64-gLuc-2A-GFP-WPREpXRP058-pLenti2-EF1a-Cas9-GGGGGS₃-NLS-SID4X-gLuc-2A-GFP-WPREpXRP059-pLenti2-EF1a-Cas9-GGGGGS₃-NLS-KRAB-gLuc-2A-GFP-WPREpXRP060-pLenti2-EF1a-Cas9-EAAAK₃-NLS-VP64-gLuc-2A-GFP-WPREpXRP061-pLenti2-EF1a-Cas9-EAAAK₃-NLS-SID4X-gLuc-2A-GFP-WPREpXRP062-pLenti2-EF1a-Cas9-EAAAK₃-NLS-KRAB-gLuc-2A-GFP-WPRE

Example 12 Targeted Deletion of Genes Involved in CholesterolBiosynthesis, Fatty Acid Biosynthesis, and Other Metabolic Disorders,Genes Encoding Mis-Folded Proteins Involved in Amyloid and OtherDiseases, Oncogenes Leading to Cellular Transformation, Latent ViralGenes, and Genes Leading to Dominant-Negative Disorders, Amongst OtherDisorders

Applicants demonstrate gene delivery of a CRISPR-Cas system in theliver, brain, ocular, epithelial, hematopoetic, or another tissue of asubject or a patient in need thereof, suffering from metabolicdisorders, amyloidosis and protein-aggregation related diseases,cellular transformation arising from genetic mutations andtranslocations, dominant negative effects of gene mutations, latentviral infections, and other related symptoms, using either viral ornanoparticle delivery system.

Study Design:

Subjects or patients in need thereof suffering from metabolic disorders,amyloidosis and protein aggregation related disease which include butare not limited to human, non-primate human, canine, feline, bovine,equine, other domestic animals and related mammals. The CRISPR-Cassystem is guided by a chimeric guide RNA and targets a specific site ofthe human genomic loci to be cleaved. After cleavage and non-homologousend-joining mediated repair, frame-shift mutation results in knock outof genes.

Applicants select guide-RNAs targeting genes involved in above-mentioneddisorders to be specific to endogenous loci with minimal off-targetactivity. Two or more guide RNAs may be encoded into a single CRISPRarray to induce simultaneous double-stranded breaks in DNA leading tomicro-deletions of affected genes or chromosomal regions.

Identification and Design of Gene Targets

For each candidate disease gene, Applicants select DNA sequences ofinterest include protein-coding exons, sequences including and flankingknown dominant negative mutation sites, sequences including and flankingpathological repetitive sequences. For gene-knockout approaches, earlycoding exons closest to the start codon offer best options for achievingcomplete knockout and minimize possibility of truncated protein productsretaining partial function.

Applicants analyze sequences of interest for all possible targetable20-bp sequences immediately 5′ to a NGG motif (for SpCas9 system) or aNNAGAAW (for StlCas9 system). Applicants choose sequences for unique,single RNA-guided Cas9 recognition in the genome to minimize off-targeteffects based on computational algorithm to determine specificity.

Cloning of guide sequences into a delivery system

Guide sequences are synthesized as double-stranded 20-24 bpoligonucleotides. After 5′-phosphorylation treatment of oligos andannealing to form duplexes, oligos are ligated into suitable vectordepending on the delivery method:

Virus-based delivery methods

AAV-based vectors (PX260, 330, 334, 335) have been described elsewhere

Lentiviral-based vectors use a similar cloning strategy of directlyligating guide sequences into a single vector carrying a U6promoter-driven chimeric RNA scaffold and a EF1a promoter-driven Cas9 orCas9 nickase.

Virus production is described elsewhere.

Nanoparticle-based RNA delivery methods

1. Guide sequences are synthesized as an oligonucleotide duplex encodingT7 promoter-guide sequence-chimeric RNA. A T7 promoter is added 5′ ofCas9 by PCR method.

2. T7-driven Cas9 and guide-chimeric RNAs are transcribed in vitro, andCas9 mRNA is further capped and A-tailed using commercial kits. RNAproducts are purified per kit instructions.

Hydrodynamic tail vein delivery methods (for mouse)

Guide sequences are cloned into AAV plasmids as described above andelsewhere in this application.

In vitro validation on cell lines

Transfection

1. DNA plasmid transfection

Plasmids carrying guide sequences are transfected into human embryonickidney (HEK293T) or human embryonic stem (hES) cells, other relevantcell types using lipid-, chemical-, or electroporation-based methods.For a 24-well transfection of HEK293T cells (˜260,000 cells), 500 ng oftotal DNA is transfected into each single well using Lipofectamine 2000.For a 12-well transfection of hES cells, 1 ug of total DNA istransfected into a single well using Fugene HD.

2. RNA transfection

Purified RNA described above is used for transfection into HEK293Tcells. 1-2 ug of RNA may be transfected into 260,000 using Lipofectamine2000 per manufacturer's instruction. RNA delivery of Cas9 and chimericRNA is shown in FIG. 10.

Assay of Indel Formation In Vitro

Cells are harvested 72-hours post-transfection and assayed for indelformation as an indication of double-stranded breaks.

Briefly, genomic region around target sequence is PCR amplified(˜400-600 bp amplicon size) using high-fidelity polymerase. Products arepurified, normalized to equal concentration, and slowly annealed from95° C. to 4° C. to allow formation of DNA heteroduplexes. Postannealing, the Cel-1 enzyme is used to cleave heteroduplexes, andresulting products are separated on a polyacrylamide gel and indelefficiency calculated.

In vivo proof of principle in animal

Delivery mechanisms

AAV or Lentivirus production is described elsewhere.

Nanoparticle formulation: RNA mixed into nanoparticle formulation

Hydrodynamic tail vein injections with DNA plasmids in mice areconducted using a commercial kit

Cas9 and guide sequences are delivered as virus, nanoparticle-coated RNAmixture, or DNA plasmids, and injected into subject animals. A parallelset of control animals is injected with sterile saline, Cas9 and GFP, orguide sequence and GFP alone.

Three weeks after injection, animals are tested for amelioration ofsymptoms and sacrificed. Relevant organ systems analyzed for indelformation. Phenotypic assays include blood levels of HDL, LDL, lipids,

Assay for Indel Formation

DNA is extracted from tissue using commercial kits; indel assay will beperformed as described for in vitro demonstration.

Therapeutic applications of the CRISPR-Cas system are amenable forachieving tissue-specific and temporally controlled targeted deletion ofcandidate disease genes. Examples include genes involved in cholesteroland fatty acid metabolism, amyloid diseases, dominant negative diseases,latent viral infections, among other disorders.

Examples of a single guide-RNA to introduce targeted indels at a genelocus

SEQ ID Disease GENE SPACER PAM NO: Mechanism References Hyperchole HMG-GCCAAA CGG 100 Knockout Fluvastatin: a review of its sterolemia CRTTGGAC pharmacology and use in the GACCCTCG management ofhypercholesterolaemia. (Plosker GL et al. Drugs 1996, 51(3): 433-459)Hyperchole SQLE CGAGGA TGG 101 Knockout Potential role of nonstatinsterolemia GACCCC cholesterol lowering agents CGTTTCGG (Trapani et al.IUBMB Life, Volume 63, Issue 11, pages 964-971, November 2011)Hyperlipidemia DGAT1 CCCGCC AGG 102 Knockout DGAT1 inhibitors as anti-GCCGCC obesity and anti-diabetic GTGGCTCG agents. (Birch AM et al.Current Opinion in Drug Discovery & Development [2010, 13(4): 489-496)Leukemia BCR- TGAGCTC AGG 103 Knockout Killing of leukemic cells ABLTACGAG with a BCR/ABL fusion ATCCACA gene by RNA interference (RNAi).(Fuchs et al. Oncogene 2002, 21(37): 5716-5724)

Examples of a pair of guide-RNA to introduce chromosomal microdeletionat a gene locus

SEQ ID Disease GENE SPACER PAM NO: Mechanism References HyperlipidemiaPLIN2 CTCAAA TGG 104 Microdeletion Perilipin-2 Null Mice are guide1ATTCATA Protected Against Diet- CCGGTTG Induced Obesity, AdiposeInflammation and Fatty Liver Disease (McManaman JL et al. The Journal ofLipid Research, jlr.M035063. First Published on Feb. 12, 2013)Hyperlipidemia PLIN2 CGTTAAA TGG 105 Microdeletion guide2 CAACAA CCGGACTHyperlipidemia SREBP TTCACCC ggg 106 Microdeletion Inhibition of SREBPby a guide1 CGCGGC Small Molecule, Betulin, GCTGAAT ImprovesHyperlipidemia and Insulin Resistance and Reduces AtheroscleroticPlaques (Tang J et al. Cell Metabolism, Volume 13, Issue 1, 44-56, 5January 2011) Hyperlipidemia SREBP ACCACTA agg 107 Microdeletion guide2CCAGTCC GTCCAC

Example 13 Targeted Integration of Repair for Genes CarryingDisease-Causing Mutations; Reconstitution of Enzyme Deficiencies andOther Related Diseases

Study Design

I. Identification and design of gene targets

-   -   Described in Example 16

II. Cloning of guide sequences and repair templates into a deliverysystem

-   -   Described above in Example 16    -   Applicants clone DNA repair templates to include homology arms        with diseased allele as well a wild-type repair template

III. In vitro validation on cell lines

-   -   a. Transfection is described above in Example 16; Cas9, guide        RNAs, and repair template are co-transfected into relevant cell        types.    -   b. Assay for repair in vitro        -   i. Applicants harvest cells 72-hours post-transfection and            assay for repair        -   ii. Briefly, Applicants amplify genomic region around repair            template PCR using high-fidelity polymerase. Applicants            sequence products for decreased incidence of mutant allele.

IV. In vivo proof of principle in animal

-   -   a. Delivery mechanisms are described above Examples 16 and 29.    -   b. Assay for repair in vivo        -   i. Applicants perform the repair assay as described in the            in vitro demonstration.

V. Therapeutic applications

-   -   The CRISPR-Cas system is amenable for achieving tissue-specific        and temporally controlled targeted deletion of candidate disease        genes. Examples include genes involved in cholesterol and fatty        acid metabolism, amyloid diseases, dominant negative diseases,        latent viral infections, among other disorders.

Example of one single missense mutation with repair template:

Disease GENE SPACER PAM Familial amyloid TTR AGCCTTTCTGAACACATGCA CGGpolyneuropathy (SEQ ID NO: 108) Mechanism References V30M repairTransthyretin mutations in health and disease (Joao et al. HumanMutation, Volume 5, Issue 3, pages 191-196, 1995) V30MCCTGCCATCAATGTGGCC

TGCATGTGTTCAGAAAGGCT allele (SEQ ID NO: 109) WT CCTGCCATCAATGTGGCC

TGCATGTGTTCAGAAAGGCT allele (SEQ ID NO: 110)

Example 14 Therapeutic Application of the CRISPR-Cas System in Glaucoma,Amyloidosis, and Huntington's Disease

Glaucoma: Applicants design guide RNAs to target the first exon of themycilin (MYOC) gene. Applicants use adenovirus vectors (Ad5) to packageboth Cas9 as well as a guide RNA targeting the MYOC gene. Applicantsinject adenoviral vectors into the trabecular meshwork where cells havebeen implicated in the pathophysiology of glaucoma. Applicants initiallytest this out in mouse models carrying the mutated MYOC gene to seewhether they improve visual acuity and decrease pressure in the eyes.Therapeutic application in humans employ a similar strategy.

Amyloidosis: Applicants design guide RNAs to target the first exon ofthe transthyretin (TTR) gene in the liver. Applicants use AAV8 topackage Cas9 as well as guide RNA targeting the first exon of the TTRgene. AAV8 has been shown to have efficient targeting of the liver andwill be administered intravenously. Cas9 can be driven either usingliver specific promoters such as the albumin promoter, or using aconstitutive promoter. A pol3 promoter drives the guide RNA.

Alternatively, Applicants utilize hydrodynamic delivery of plasmid DNAto knockout the TTR gene. Applicants deliver a plasmid encoding Cas9 andthe guideRNA targeting Exonl of TTR.

As a further alternative approach, Applicants administer a combinationof RNA (mRNA for Cas9, and guide RNA). RNA can be packaged usingliposomes such as Invivofectamine from Life Technologies and deliveredintravenously. To reduce RNA-induced immunogenicity, increase the levelof Cas9 expression and guide RNA stability, Applicants modify the Cas9mRNA using 5′ capping. Applicants also incorporate modified RNAnucleotides into Cas9 mRNA and guide RNA to increase their stability andreduce immunogenicity (e.g. activation of TLR). To increase efficiency,Applicants administer multiple doses of the virus, DNA, or RNA.

Huntington's Disease: Applicants design guide RNA based on allelespecific mutations in the HTT gene of patients. For example, in apatient who is heterozygous for HTT with expanded CAG repeat, Applicantsidentify nucleotide sequences unique to the mutant HTT allele and use itto design guideRNA. Applicants ensure that the mutant base is locatedwithin the last 9 bp of the guide RNA (which Applicants have ascertainedhas the ability to discriminate between single DNA base mismatchesbetween the target size and the guide RNA).

Applicants package the mutant HTT allele specific guide RNA and Cas9into AAV9 and deliver into the striatum of Huntington's patients. Virusis injected into the striatum stereotactically via a craniotomy. AAV9 isknown to transduce neurons efficiently. Applicants drive Cas9 using aneuron specific promoter such as human Synapsin I.

Example 15 Therapeutic Application of the CRISPR-Cas System in HIV

Chronic viral infection is a source of significant morbidity andmortality. While there exists for many of these viruses conventionalantiviral therapies that effectively target various aspects of viralreplication, current therapeutic modalities are usually non-curative innature due to “viral latency.” By its nature, viral latency ischaracterized by a dormant phase in the viral life cycle without activeviral production. During this period, the virus is largely able to evadeboth immune surveillance and conventional therapeutics allowing for itto establish long-standing viral reservoirs within the host from whichsubsequent re-activation can permit continued propagation andtransmission of virus. Key to viral latency is the ability to stablymaintain the viral genome, accomplished either through episomal orproviral latency, which stores the viral genome in the cytoplasm orintegrates it into the host genome, respectively. In the absence ofeffective vaccinations which would prevent primary infection, chronicviral infections characterized by latent reservoirs and episodes oflytic activity can have significant consequences: human papilloma virus(HPV) can result in cervical cancer, hepatitis C virus (HCV) predisposesto hepatocellular carcinoma, and human immunodeficiency virus eventuallydestroys the host immune system resulting in susceptibility toopportunistic infections. As such, these infections require life-longuse of currently available antiviral therapeutics. Further complicatingmatters is the high mutability of many of these viral genomes which leadto the evolution of resistant strains for which there exists noeffective therapy.

The CRISPR-Cas system is a bacterial adaptive immune system able toinduce double-stranded DNA breaks (DSB) in a multiplex-able,sequence-specific manner and has been recently re-constituted withinmammalian cell systems. It has been shown that targeting DNA with one ornumerous guide-RNAs can result in both indels and deletions of theintervening sequences, respectively. As such, this new technologyrepresents a means by which targeted and multiplexed DNA mutagenesis canbe accomplished within a single cell with high efficiency andspecificity. Consequently, delivery of the CRISPR-Cas system directedagainst viral DNA sequences could allow for targeted disruption anddeletion of latent viral genomes even in the absence of ongoing viralproduction.

As an example, chronic infection by HIV-1 represents a global healthissue with 33 million individuals infected and an annual incidence of2.6 million infections. The use of the multimodal highly activeantiretroviral therapy (HAART), which simultaneously targets multipleaspects of viral replication, has allowed HIV infection to be largelymanaged as a chronic, not terminal, illness. Without treatment,progression of HIV to AIDS occurs usually within 9-10 years resulting indepletion of the host immune system and occurrence of opportunisticinfections usually leading to death soon thereafter. Secondary to virallatency, discontinuation of HAART invariably leads to viral rebound.Moreover, even temporary disruptions in therapy can select for resistantstrains of HIV uncontrollable by available means. Additionally, thecosts of HAART therapy are significant: within the US $10,000-15,0000per person per year. As such, treatment approaches directly targetingthe HIV genome rather than the process of viral replication represents ameans by which eradication of latent reservoirs could allow for acurative therapeutic option.

Development and delivery of an HIV-1 targeted CRISPR-Cas systemrepresents a unique approach differentiable from existing means oftargeted DNA mutagenesis, i.e. ZFN and TALENs, with numerous therapeuticimplications. Targeted disruption and deletion of the HIV-1 genome byCRISPR-mediated DSB and indels in conjunction with HAART could allow forsimultaneous prevention of active viral production as well as depletionof latent viral reservoirs within the host.

Once integrated within the host immune system, the CRISPR-Cas systemallows for generation of a HIV-1 resistant sub-population that, even inthe absence of complete viral eradication, could allow for maintenanceand re-constitution of host immune activity. This could potentiallyprevent primary infection by disruption of the viral genome preventingviral production and integration, representing a means to “vaccination”.Multiplexed nature of the CRISPR-Cas system allows targeting of multipleaspects of the genome simultaneously within individual cells.

As in HAART, viral escape by mutagenesis is minimized by requiringacquisition of multiple adaptive mutations concurrently. Multiplestrains of HIV-1 can be targeted simultaneously which minimizes thechance of super-infection and prevents subsequent creation of newrecombinants strains. Nucleotide, rather than protein, mediatedsequence-specificity of the CRISPR-Cas system allows for rapidgeneration of therapeutics without need for significantly alteringdelivery mechanism.

In order to accomplish this, Applicants generate CRISPR-Cas guide RNAsthat target the vast majority of the HIV-1 genome while taking intoaccount HIV-1 strain variants for maximal coverage and effectiveness.Sequence analyses of genomic conservation between HIV-1 subtypes andvariants should allow for targeting of flanking conserved regions of thegenome with the aims of deleting intervening viral sequences orinduction of frame-shift mutations which would disrupt viral genefunctions.

Applicants accomplish delivery of the CRISPR-Cas system by conventionaladenoviral or lentiviral-mediated infection of the host immune system.Depending on approach, host immune cells could be a) isolated,transduced with CRISPR-Cas, selected, and re-introduced in to the hostor b) transduced in vivo by systemic delivery of the CRISPR-Cas system.The first approach allows for generation of a resistant immunepopulation whereas the second is more likely to target latent viralreservoirs within the host.

Examples of potential HIV-1 targeted spacers adapted from Mcintyre etal, which generated shRNAs against HIV-1 optimized for maximal coverageof HIV-1 variants. (SEQ ID NO: 111) CACTGCTTAAGCCTCGCTCGAGG (SEQ ID NO:112) TCACCAGCAATATTCGCTCGAGG (SEQ ID NO: 113) CACCAGCAATATTCCGCTCGAGG(SEQ ID NO: 114) TAGCAACAGACATACGCTCGAGG (SEQ ID NO: 115)GGGCAGTAGTAATACGCTCGAGG (SEQ ID NO: 116) CCAATTCCCATACATTATTGTAC

Example 16 Targeted Correction of deltaF508 or Other Mutations in CysticFibrosis

An aspect of the invention provides for a pharmaceutical compositionthat may comprise an CRISPR-Cas gene therapy particle and abiocompatible pharmaceutical carrier. According to another aspect, amethod of gene therapy for the treatment of a subject having a mutationin the CFTR gene comprises administering a therapeutically effectiveamount of a CRISPR-Cas gene therapy particle to the cells of a subject.

This Example demonstrates gene transfer or gene delivery of a CRISPR-Cassystem in airways of subject or a patient in need thereof, sufferingfrom cystic fibrosis or from cystic fibrosis related symptoms, usingadeno-associated virus (AAV) particles.

Study Design: Subjects or patients in need there of: Human, non-primatehuman, canine, feline, bovine, equine and other domestic animals,related. This study tests efficacy of gene transfer of a CRISPR-Cassystem by a AAV vector. Applicants determine transgene levels sufficientfor gene expression and utilize a CRISPR-Cas system comprising a Cas9enzyme to target deltaF508 or other CFTR-inducing mutations.

The treated subjects receive pharmaceutically effective amount ofaerosolized AAV vector system per lung endobronchially delivered whilespontaneously breathing. The control subjects receive equivalent amountof a pseudotyped AAV vector system with an internal control gene. Thevector system may be delivered along with a pharmaceutically acceptableor biocompatible pharmaceutical carrier. Three weeks or an appropriatetime interval following vector administration, treated subjects aretested for amelioration of cystic fibrosis related symptoms.

Applicants use an adenovirus or an AAV particle.

Applicants clone the following gene constructs, each operably linked toone or more regulatory sequences (Cbh or EF1a promoter for Cas9, U6 orH1 promoter for chimeric guide RNA), into one or more adenovirus or AAVvectors or any other compatible vector: A CFTRdelta508 targetingchimeric guide RNA (FIG. 13B), a repair template for deltaF508 mutation(FIG. 13C) and a codon optimized Cas9 enzyme with optionally one or morenuclear localization signal or sequence(s) (NLS(s)), e.g., two (2) NLSs.

Identification of Cas9 Target Site

Applicants analyzed the human CFTR genomic locus and identified the Cas9target site (FIG. 13A). (PAM may contain a NGG or a NNAGAAW motif).

Gene Repair Strategy

Applicants introduce an adenovirus/AAV vector system comprising a Cas9(or Cas9 nickase) and the guide RNA along with a adenovirus/AAV vectorsystem comprising the homology repair template containing the F508residue into the subject via one of the methods of delivery discussedearlier. The CRISPR-Cas system is guided by the CFTRdelta 508 chimericguide RNA and targets a specific site of the CFTR genomic locus to benicked or cleaved. After cleavage, the repair template is inserted intothe cleavage site via homologous recombination correcting the deletionthat results in cystic fibrosis or causes cystic fibrosis relatedsymptoms. This strategy to direct delivery and provide systemicintroduction of CRISPR systems with appropriate guide RNAs can beemployed to target genetic mutations to edit or otherwise manipulategenes that cause metabolic, liver, kidney and protein diseases anddisorders such as those in Table B.

Example 17 Generation of Gene Knockout Cell Library

This example demonstrates how to generate a library of cells where eachcell has a single gene knocked out:

Applicants make a library of ES cells where each cell has a single geneknocked out, and the entire library of ES cells will have every singlegene knocked out. This library is useful for the screening of genefunction in cellular processes as well as diseases.

To make this cell library, Applicants integrate Cas9 driven by aninducible promoter (e.g. doxycycline inducible promoter) into the EScell. In addition, Applicants integrate a single guide RNA targeting aspecific gene in the ES cell. To make the ES cell library, Applicantssimply mix ES cells with a library of genes encoding guide RNAstargeting each gene in the human genome. Applicants first introduce asingle BxB1 attB site into the AAVS1 locus of the human ES cell. ThenApplicants use the BxB1 integrase to facilitate the integration ofindividual guide RNA genes into the BxB1 attB site in AAVS1 locus. Tofacilitate integration, each guide RNA gene is contained on a plasmidthat carries of a single attP site. This way BxB1 will recombine theattB site in the genome with the attP site on the guide RNA containingplasmid.

To generate the cell library, Applicants take the library of cells thathave single guide RNAs integrated and induce Cas9 expression. Afterinduction, Cas9 mediates double strand break at sites specified by theguide RNA. To verify the diversity of this cell library, Applicantscarry out whole exome sequencing to ensure that Applicants are able toobserve mutations in every single targeted gene. This cell library canbe used for a variety of applications, including who library-basedscreens, or can be sorted into individual cell clones to facilitaterapid generation of clonal cell lines with individual human genesknocked out.

Example 18 Engineering of Microalgae Using Cas9

Methods of Delivering Cas9

Method 1: Applicants deliver Cas9 and guide RNA using a vector thatexpresses Cas9 under the control of a constitutive promoter such asHsp70A-Rbc S2 or Beta2-tubulin.

Method 2: Applicants deliver Cas9 and T7 polymerase using vectors thatexpresses Cas9 and T7 polymerase under the control of a constitutivepromoter such as Hsp70A-Rbc S2 or Beta2-tubulin. Guide RNA will bedelivered using a vector containing T7 promoter driving the guide RNA.

Method 3: Applicants deliver Cas9 mRNA and in vitro transcribed guideRNA to algae cells. RNA can be in vitro transcribed. Cas9 mRNA willconsist of the coding region for Cas9 as well as 3′UTR from Cop1 toensure stabilization of the Cas9 mRNA.

For Homologous recombination, Applicants provide an additional homologydirected repair template.

Sequence for a cassette driving the expression of Cas9 under the controlof beta-2 tubulin promoter, followed by the 3′ UTR of Cop1.

(SEQ ID NO: 117) TCTTTCTTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCAACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCACTACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAACATGTACCCATACGATGTTCCAGATTACGCTTCGCCGAAGAAAAAGCGCAAGGTCGAAGCGTCCGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGCAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGAGCCACTCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACAGCCCCAAGAAGAAGAGAAAGGTGGAGGCCAGCTAAGGATCCGGCAAGACTGGCCCCGCTTGGCAACGCAACAGTGAGCCCCTCCCTAGTGTGTTTGGGGATGTGACTATGTATTCGTGTGTTGGCCAACGGGTCAACCCGAACAGATTGATACCCGCCTTGGCATTTCCTGTCAGAATGTAACGTCAGTTGATGGTACT

Sequence for a cassette driving the expression of T7 polymerase underthe control of beta-2 tubulin promoter, followed by the 3′ UTR of Cop1:

(SEQ ID NO: 118) TCTTTCTTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCAACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCACTACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAACatgcctaagaagaagaggaaggttaacacgattaacatcgctaagaacgacttctctgacatcgaactggctgctatcccgttcaacactctggctgaccattacggtgagcgtttagctcgcgaacagttggcccttgagcatgagtcttacgagatgggtgaagcacgcttccgcaagatgtttgagcgtcaacttaaagctggtgaggttgcggataacgctgccgccaagcctctcatcactaccctactccctaagatgattgcacgcatcaacgactggtttgaggaagtgaaagctaagcgcggcaagcgcccgacagccttccagttcctgcaagaaatcaagccggaagccgtagcgtacatcaccattaagaccactctggcttgcctaaccagtgctgacaatacaaccgttcaggctgtagcaagcgcaatcggtcgggccattgaggacgaggctcgcttcggtcgtatccgtgaccttgaagctaagcacttcaagaaaaacgttgaggaacaactcaacaagcgcgtagggcacgtctacaagaaagcatttatgcaagttgtcgaggctgacatgctctctaagggtctactcggtggcgaggcgtggtcttcgtggcataaggaagactctattcatgtaggagtacgctgcatcgagatgctcattgagtcaaccggaatggttagcttacaccgccaaaatgctggcgtagtaggtcaagactctgagactatcgaactcgcacctgaatacgctgaggctatcgcaacccgtgcaggtgcgctggctggcatctctccgatgttccaaccttgcgtagttcctcctaagccgtggactggcattactggtggtggctattgggctaacggtcgtcgtcctctggcgctggtgcgtactcacagtaagaaagcactgatgcgctacgaagacgtttacatgcctgaggtgtacaaagcgattaacattgcgcaaaacaccgcatggaaaatcaacaagaaagtcctagcggtcgccaacgtaatcaccaagtggaagcattgtccggtcgaggacatccctgcgattgagcgtgaagaactcccgatgaaaccggaagacatcgacatgaatcctgaggctctcaccgcgtggaaacgtgctgccgctgctgtgtaccgcaaggacaaggctcgcaagtctcgccgtatcagccttgagttcatgcttgagcaagccaataagtttgctaaccataaggccatctggttcccttacaacatggactggcgcggtcgtgtttacgctgtgtcaatgttcaacccgcaaggtaacgatatgaccaaaggactgcttacgctggcgaaaggtaaaccaatcggtaaggaaggttactactggctgaaaatccacggtgcaaactgtgcgggtgtcgacaaggttccgttccctgagcgcatcaagttcattgaggaaaaccacgagaacatcatggcttgcgctaagtctccactggagaacacttggtgggctgagcaagattctccgttctgcttccttgcgttctgctttgagtacgctggggtacagcaccacggcctgagctataactgctcccttccgctggcgtttgacgggtcttgctctggcatccagcacttctccgcgatgctccgagatgaggtaggtggtcgcgcggttaacttgcttcctagtgaaaccgttcaggacatctacgggattgttgctaagaaagtcaacgagattctacaagcagacgcaatcaatgggaccgataacgaagtagttaccgtgaccgatgagaacactggtgaaatctctgagaaagtcaagctgggcactaaggcactggctggtcaatggctggcttacggtgttactcgcagtgtgactaagcgttcagtcatgacgctggcttacgggtccaaagagttcggcttccgtcaacaagtgctggaagataccattcagccagctattgattccggcaagggtctgatgttcactcagccgaatcaggctgctggatacatggctaagctgatttgggaatctgtgagcgtgacggtggtagctgcggttgaagcaatgaactggcttaagtctgctgctaagctgctggctgctgaggtcaaagataagaagactggagagattcttcgcaagcgttgcgctgtgcattgggtaactcctgatggtttccctgtgtggcaggaatacaagaagcctattcagacgcgcttgaacctgatgttcctcggtcagttccgcttacagcctaccattaacaccaacaaagatagcgagattgatgcacacaaacaggagtctggtatcgctcctaactttgtacacagccaagacggtagccaccttcgtaagactgtagtgtgggcacacgagaagtacggaatcgaatcttttgcactgattcacgactccttcggtacgattccggctgacgctgcgaacctgttcaaagcagtgcgcgaaactatggttgacacatatgagtcttgtgatgtactggctgatttctacgaccagttcgctgaccagttgcacgagtctcaattggacaaaatgccagcacttccggctaaaggtaacttgaacctccgtgacatcttagagtcggacttcgcgttcgcgtaaGGATCCGGCAAGACTGGCCCCGCTTGGCAACGCAACAGTGAGCCCCTCCCTAGTGTGTTTGGGGATGTGACTATGTATTCGTGTGTTGGCCAACGGGTCAACCCGAACAGATTGATACCCGCCTTGGCATTTCCTGTCAGAATGTAACGTCAGTTGATGGTA CT

Sequence of guide RNA driven by the T7 promoter (T7 promoter, Nsrepresent targeting sequence):

(SEQ ID NO: 119) gaaatTAATACGACTCACTATA NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAAtagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcttttttt

Gene Delivery:

Chlamydomonas reinhardtii strain CC-124 and CC-125 from theChlamydomonas Resource Center will be used for electroporation.Electroporation protocol follows standard recommended protocol from theGeneArt Chlamydomonas Engineering kit.

Also, Applicants generate a line of Chlamydomonas reinhardtii thatexpresses Cas9 constitutively. This can be done by using pChlamy1(linearized using PvuI) and selecting for hygromycin resistant colonies.Sequence for pChlamy1 containing Cas9 is below. In this way to achievegene knockout one simply needs to deliver RNA for the guideRNA. Forhomologous recombination Applicants deliver guideRNA as well as alinearized homologous recombination template.

pChlamy1-Cas9:

(SEQ ID NO: 120) TGCGGTATTTCACACCGCATCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGTTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGTCGCTGAGGCTTGACATGATTGGTGCGTATGTTTGTATGAAGCTACAGGACTGATTTGGCGGGCTATGAGGGCGGGGGAAGCTCTGGAAGGGCCGCGATGGGGCGCGCGGCGTCCAGAAGGCGCCATACGGCCCGCTGGCGGCACCCATCCGGTATAAAAGCCCGCGACCCCGAACGGTGACCTCCACTTTCAGCGACAAACGAGCACTTATACATACGCGACTATTCTGCCGCTATACATAACCACTCAGCTAGCTTAAGATCCCATCAAGCTTGCATGCCGGGCGCGCCAGAAGGAGCGCAGCCAAACCAGGATGATGTTTGATGGGGTATTTGAGCACTTGCAACCCTTATCCGGAAGCCCCCTGGCCCACAAAGGCTAGGCGCCAATGCAAGCAGTTCGCATGCAGCCCCTGGAGCGGTGCCCTCCTGATAAACCGGCCAGGGGGCCTATGTTCTTTACTTTTTTACAAGAGAAGTCACTCAACATCTTAAAATGGCCAGGTGAGTCGACGAGCAAGCCCGGCGGATCAGGCAGCGTGCTTGCAGATTTGACTTGCAACGCCCGCATTGTGTCGACGAAGGCTTTTGGCTCCTCTGTCGCTGTCTCAAGCAGCATCTAACCCTGCGTCGCCGTTTCCATTTGCAGGAGATTCGAGGTACCATGTACCCATACGATGTTCCAGATTACGCTTCGCCGAAGAAAAAGCGCAAGGTCGAAGCGTCCGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGCAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACAGCCCCAAGAAGAAGAGAAAGGTGGAGGCCAGCTAACATATGATTCGAATGTCTTTCTTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCAACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCACTACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAACATGACACAAGAATCCCTGTTACTTCTCGACCGTATTGATTCGGATGATTCCTACGCGAGCCTGCGGAACGACCAGGAATTCTGGGAGGTGAGTCGACGAGCAAGCCCGGCGGATCAGGCAGCGTGCTTGCAGATTTGACTTGCAACGCCCGCATTGTGTCGACGAAGGCTTTTGGCTCCTCTGTCGCTGTCTCAAGCAGCATCTAACCCTGCGTCGCCGTTTCCATTTGCAGCCGCTGGCCCGCCGAGCCCTGGAGGAGCTCGGGCTGCCGGTGCCGCCGGTGCTGCGGGTGCCCGGCGAGAGCACCAACCCCGTACTGGTCGGCGAGCCCGGCCCGGTGATCAAGCTGTTCGGCGAGCACTGGTGCGGTCCGGAGAGCCTCGCGTCGGAGTCGGAGGCGTACGCGGTCCTGGCGGACGCCCCGGTGCCGGTGCCCCGCCTCCTCGGCCGCGGCGAGCTGCGGCCCGGCACCGGAGCCTGGCCGTGGCCCTACCTGGTGATGAGCCGGATGACCGGCACCACCTGGCGGTCCGCGATGGACGGCACGACCGACCGGAACGCGCTGCTCGCCCTGGCCCGCGAACTCGGCCGGGTGCTCGGCCGGCTGCACAGGGTGCCGCTGACCGGGAACACCGTGCTCACCCCCCATTCCGAGGTCTTCCCGGAACTGCTGCGGGAACGCCGCGCGGCGACCGTCGAGGACCACCGCGGGTGGGGCTACCTCTCGCCCCGGCTGCTGGACCGCCTGGAGGACTGGCTGCCGGACGTGGACACGCTGCTGGCCGGCCGCGAACCCCGGTTCGTCCACGGCGACCTGCACGGGACCAACATCTTCGTGGACCTGGCCGCGACCGAGGTCACCGGGATCGTCGACTTCACCGACGTCTATGCGGGAGACTCCCGCTACAGCCTGGTGCAACTGCATCTCAACGCCTTCCGGGGCGACCGCGAGATCCTGGCCGCGCTGCTCGACGGGGCGCAGTGGAAGCGGACCGAGGACTTCGCCCGCGAACTGCTCGCCTTCACCTTCCTGCACGACTTCGAGGTGTTCGAGGAGACCCCGCTGGATCTCTCCGGCTTCACCGATCCGGAGGAACTGGCGCAGTTCCTCTGGGGGCCGCCGGACACCGCCCCCGGCGCCTGATAAGGATCCGGCAAGACTGGCCCCGCTTGGCAACGCAACAGTGAGCCCCTCCCTAGTGTGTTTGGGGATGTGACTATGTATTCGTGTGTTGGCCAACGGGTCAACCCGAACAGATTGATACCCGCCTTGGCATTTCCTGTCAGAATGTAACGTCAGTTGATG GTACT

For all modified Chlamydomonas reinhardtii cells, Applicants use PCR,SURVEYOR nuclease assay, and DNA sequencing to verify successfulmodification.

Example 19 Use of Cas9 to Target a Variety of Disease Types

Diseases that involve mutations in protein coding sequence:

Dominant disorders may be targeted by inactivating the dominant negativeallele. Applicants use Cas9 to target a unique sequence in the dominantnegative allele and introduce a mutation via NHEJ. The NHEJ-inducedindel may be able to introduce a frame-shift mutation in the dominantnegative allele and eliminate the dominant negative protein. This maywork if the gene is haplo-sufficient (e.g. MYOC mutation inducedglaucoma and Huntington's disease).

Recessive disorders may be targeted by repairing the disease mutation inboth alleles. For dividing cells, Applicants use Cas9 to introducedouble strand breaks near the mutation site and increase the rate ofhomologous recombination using an exogenous recombination template. Fordividing cells, this may be achieved using multiplexed nickase activityto catalyze the replacement of the mutant sequence in both alleles viaNHEJ-mediated ligation of an exogenous DNA fragment carryingcomplementary overhangs.

Applicants also use Cas9 to introduce protective mutations (e.g.inactivation of CCR5 to prevent HIV infection, inactivation of PCSK9 forcholesterol reduction, or introduction of the A673T into APP to reducethe likelihood of Alzheimer's disease).

Diseases that Involve Non-Coding Sequences

Applicants use Cas9 to disrupt non-coding sequences in the promoterregion, to alter transcription factor binding sites and alter enhanceror repressor elements. For example, Cas9 may be used to excise out theKlf1 enhancer EHS1 in hematopoietic stem cells to reduce BCL11a levelsand reactivate fetal globin gene expression in differentiatederythrocytes

Applicants also use Cas9 to disrupt functional motifs in the 5′ or 3′untranslated regions. For example, for the treatment of myotonicdystrophy, Cas9 may be used to remove CTG repeat expansions in the DMPKgene.

Example 20 Multiplexed Nickase

Aspects of optimization and the teachings of Cas9 detailed in thisapplication may also be used to generate Cas9 nickases. Applicants useCas9 nickases in combination with pairs of guide RNAs to generate DNAdouble strand breaks with defined overhangs. When two pairs of guideRNAs are used, it is possible to excise an intervening DNA fragment. Ifan exogenous piece of DNA is cleaved by the two pairs of guide RNAs togenerate compatible overhangs with the genomic DNA, then the exogenousDNA fragment may be ligated into the genomic DNA to replace the excisedfragment. For example, this may be used to remove trinucleotide repeatexpansion in the huntintin (HTT) gene to treat Huntington's Disease.

If an exogenous DNA that bears fewer number of CAG repeats is provided,then it may be able to generate a fragment of DNA that bears the sameoverhangs and can be ligated into the HTT genomic locus and replace theexcised fragment (fragments below disclosed as SEQ ID NOS 121-128,respectively, in order of appearance).

HTT locus with ...CCGTGCCGGGCGGGAGACCGCCATGG GGCCCGGCTGTGGCTGAGGAGC...fragment ...GGCACGGCCCGCCCTCTGGC TGGGCCGGGCCGACACCGACTCCTCG... excisedby Cas9 nickase and two pairs of guide RNAs + exogenous DNACGACCCTGGAAA..... reduced number of CAG repeats .....CCCCGCCGCCACCCfragment with GGTACCGCTGGGACCTTT..... .....GGGGCGGCGG fewer number ofCAG repeats also cleaved by Cas9 nicakse and the two pairs of guide RNAs

The ligation of the exogenous DNA fragment into the genome does notrequire homologous recombination machineries and therefore this methodmay be used in post-mitotic cells such as neurons.

Example 21 Delivery of CRISPR System

Cas9 and its chimeric guide RNA, or combination of tracrRNA and crRNA,can be delivered either as DNA or RNA. Delivery of Cas9 and guide RNAboth as RNA (normal or containing base or backbone modifications)molecules can be used to reduce the amount of time that Cas9 proteinpersist in the cell. This may reduce the level of off-target cleavageactivity in the target cell. Since delivery of Cas9 as mRNA takes timeto be translated into protein, it might be advantageous to deliver theguide RNA several hours following the delivery of Cas9 mRNA, to maximizethe level of guide RNA available for interaction with Cas9 protein.

In situations where guide RNA amount is limiting, it may be desirable tointroduce Cas9 as mRNA and guide RNA in the form of a DNA expressioncassette with a promoter driving the expression of the guide RNA. Thisway the amount of guide RNA available will be amplified viatranscription.

A variety of delivery systems can be introduced to introduce Cas9 (DNAor RNA) and guide RNA (DNA or RNA) into the host cell. These include theuse of liposomes, viral vectors, electroporation, nanoparticles,nanowires (Shalek et al., Nano Letters, 2012), exosomes. Moleculartrojan horses liposomes (Pardridge et al., Cold Spring Harb Protoc;2010; doi:10.1101/pdb.prot5407) may be used to deliver Cas9 and guideRNA across the blood brain barrier.

Example 22 Cas9 Orthologs

Applicants analyzed Cas9 orthologs (FIGS. 3 and 4A-F) to identify therelevant PAM sequences and the corresponding chimeric guide RNAs. Thisexpanded set of PAMs may provide broader targeting across the genome andalso significantly increases the number of unique target sites andprovides potential for identifying novel Cas9s with increased levels ofspecificity in the genome. Applicants determined the PAM forStaphylococcus aureus sp. Aureus Cas9 to be NNGRR Staphylococcus aureussp. Aureus Cas9 is also known as SaCas9.

The specificity of Cas9 orthologs can be evaluated by testing theability of each Cas9 to tolerate mismatches between the guide RNA andits DNA target. For example, the specificity of SpCas9 has beencharacterized by testing the effect of mutations in the guide RNA oncleavage dficiency. Libraries of guide RNAs were made with single ormultiple mismatches between the guide sequence and the target DNA. Basedon these findings, target sites for SpCas9 can be selected based on thefollowing guidelines:

To maximize SpCas9 specificity for editing a particular gene, one shouldchoose a target site within the locus of interest such that potential‘off-target’ genomic sequences abide by the following four constraints:First and foremost, they should not be followed by a PAM with either5′-NGG or NAG sequences. Second, their global sequence similarity to thetarget sequence should be minimized. Third, a maximal number ofmismatches should lie within the PAM-proximal region of the off-targetsite. Finally, a maximal number of mismatches should be consecutive orspaced less than four bases apart.

Similar methods can be used to evaluate the specificity of other Cas9orthologs and to establish criteria for the selection of specific targetsites within the genomes of target species.

Example 23 Therapeutic Strategies for Trinucleotide Repeat Disorders

As previously mentioned in the application, the target polynucleotide ofa CRISPR complex may include a number of disease-associated genes andpolynucleotides and some of these disease associated gene may belong toa set of genetic disorders referred to as Trinucleotide repeat disorders(referred to as also trinucleotide repeat expansion disorders, tripletrepeat expansion disorders or codon reiteration disorders).

These diseases are caused by mutations in which the trinucleotiderepeats of certain genes exceed the normal, stable threshold which mayusually differ in a gene. The discovery of more repeat expansiondisorders has allowed for the classification of these disorders into anumber of categories based on underlying similar characteristics.Huntington's disease (HD) and the spinocerebellar ataxias that arecaused by a CAG repeat expansion in protein-coding portions of specificgenes are included in Category I. Diseases or disorders with expansionsthat tend to make them phenotypically diverse and include expansions areusually small in magnitude and also found in exons of genes are includedin Category II. Category III includes disorders or diseases which arecharacterized by much larger repeat expansions than either Category I orII and are generally located outside protein coding regions. Examples ofCategory III diseases or disorders include but are not limited toFragile X syndrome, myotonic dystrophy, two of the spinocerebellarataxias, juvenile myoclonic epilepsy, and Friedreich's ataxia.

Similar therapeutic strategies, like the one mentioned for Friedreich'sataxia below may be adopted to address other trinucleotide repeat orexpansion disorders as well. For example, another triple repeat diseasethat can be treated using almost identical strategy is dystrophiamyotonica 1 (DMI), where there is an expanded CTG motif in the 3′ UM. InFriedreich's ataxia, the disease results from expansion of GAAtrinucleotides in the first intron of frataxin (FXN). One therapeuticstrategy using CRISPR is to excise the GAA repeat from the first intron.The expanded GAA repeat is thought to affect the DNA structure and leadsto recruit the formation of heterochromatin which turn off the frataxingene (FIG. 14A).

Competitive Advantage Over Other Therapeutic Strategies are ListedBelow:

siRNA knockdown is not applicable in this case, as disease is due toreduced expression of frataxin. Viral gene therapy is currently beingexplored. HSV-1 based vectors were used to deliver the frataxin gene inanimal models and have shown therapeutic effect. However, long termefficacy of virus-based frataxin delivery suffer from several problems:First, it is difficult to regulate the expression of frataxin to matchnatural levels in health individuals, and second, long term overexpression of frataxin leads to cell death.

Nucleases may be used to excise the GAA repeat to restore healthygenotype, but Zinc Finger Nuclease and TAIL:EN strategies requiredelivery of two pairs of high efficacy nucleases, which is difficult forboth delivery as well as nuclease engineering (efficient excision ofgenomic DNA by ZFN or TAT EN is difficult to achieve)

In contrast to above strategies, the CRISPR-Cas system has clearadvantages. The Cas9 enzyme is more efficient and more inultiplexible,by which it is meant that one or more targets can be set at the sametime. So far, efficient excision of genomic DNA>30% by Cas9 in humancells and may be as high as 30%, and may be improved in the future.Furthermore, with regard to certain trinucleoti de repeat disorders likeHuntington's disease (HD), trinucleotide repeats in the coding regionmay be addressed if there are differences between the two alleles.Specifically, if a HD patient is heterozygous for mutant HTT and thereare nucleotide differences such as SNPs between the wt and mutant HITalleles, then Cas9 may be used to specifically target the mutant HTTallele. ZFN or TALENs will not have the ability to distinguish twoalleles based on single base differences.

In adopting a strategy using the CRISPR-Cas 9 enzyme to addressFriedreich's ataxia, Applicants design a number of guide RNAs targetingsites flanking the GAA expansion and the most efficient and specificones are chosen (FIG. 14B).

Applicants deliver a combination of guide RNAs targeting the intron I ofFXN along with Cas9 to mediate excision of the GAA expansion region.AAV9 may be used to mediate efficient delivery of Cas9 and in the spinalcord.

If the Alu element adjacent to the GAA expansion is consideredimportant, there may be constraints to the number of sites that can betargeted but Applicants may adopt strategies to avoid disrupting it.

Alternative Strategies:

Rather than modifying the genome using Cas9, Applicants may alsodirectly activate the FAN gene using Cas9 (nuclease activitydeficient)-based DNA binding domain to target a transcription activationdomain to the FXN gene. Applicants may have to address the robustness ofthe Cas9-mediated artificial transcription activation to ensure that itis robust enough as compared to other methods (Tremblay et al.;Transcription Activator-Like Effector Proteins Induce the Expression ofthe Frataxin Gene; Human Gene Therapy. August 2012, 23(8): 883-890.)

Example 24 Strategies for Minimizing Off-Target Cleavage Using Cas9Nickase

As previously mentioned in the application, Cas9 may be mutated tomediate single strand cleavage via one or more of the followingmutations: D10A, E762A, and H840A.

To mediate gene knockout via NHEJ, Applicants use a nickase version ofCas9 along with two guide RNAs. Off-target nicking by each individualguide RNA may be primarily repaired without mutation, double strandbreaks (which can lead to mutations via NHEJ) only occur when the targetsites are adjacent to each other. Since double strand breaks introducedby double nicking are not blunt, co-expression of end-processing enzymessuch as TREX1 will increase the level of NHEJ activity.

The following list of targets in tabular form are for genes involved inthe following diseases:

Lafora's Disease—target GSY1 or PPP 1R3C (PTG) to reduce glycogen inneurons.

Hypercholesterolemia—target PCSK9

Target sequences are listed in pairs (L and R) with different number ofnucleotides in the spacer (0 to 3 bp). Each spacer may also be used byitself with the wild type Cas9 to introduce double strand break at thetarget locus.

GYS1 (human) (SEQ ID NO: 129) GGCC-L ACCCTTGTTAGCCACCTCCC (SEQ ID NO:130) GGCC-R GAACGCAGTGCTCTTCGAAG (SEQ ID NO: 131) GGNCC-LCTCACGCCCTGCTCCGTGTA (SEQ ID NO: 132) GGNCC-R GGCGACAACTACTTCCTGGT (SEQID NO: 133) GGNNCC-L CTCACGCCCTGCTCCGTGTA (SEQ ID NO: 134) GGNNCC-RGGGCGACAACTACTTCCTGG (SEQ ID NO: 135) GGNNNCC-L CCTCTTCAGGGCCGGGGTGG(SEQ ID NO: 136) GGNNNCC-R GAGGACCCAGGTGGAACTGC PCSK9 (human) (SEQ IDNO: 137) GGCC-L TCAGCTCCAGGCGGTCCTGG (SEQ ID NO: 138) GGCC-RAGCAGCAGCAGCAGTGGCAG (SEQ ID NO: 139) GGNCC-L TGGGCACCGTCAGCTCCAGG (SEQID NO: 140) GGNCC-R CAGCAGTGGCAGCGGCCACC (SEQ ID NO: 141) GGNNCC-LACCTCTCCCCTGGCCCTCAT (SEQ ID NO: 142) GGNNCC-R CCAGGACCGCCTGGAGCTGA (SEQID NO: 143) GGNNNCC-L CCGTCAGCTCCAGGCGGTCC (SEQ ID NO: 144) GGNNNCC-RAGCAGCAGCAGCAGTGGCAG (human) PPP1R3C (PTG) (SEQ ID NO: 145) GGCC-LATGTGCCAAGCAAAGCCTCA (SEQ ID NO: 146) GGCC-R TTCGGTCATGCCCGTGGATG (SEQID NO: 147) GGNCC-L GTCGTTGAAATTCATCGTAC (SEQ ID NO: 148) GGNCC-RACCACCTGTGAAGAGTTTCC (SEQ ID NO: 149) GGNNCC-L CGTCGTTGAAATTCATCGTA (SEQID NO: 150) GGNNCC-R ACCACCTGTGAAGAGTTTCC Gys1 (mouse) (SEQ ID NO: 151)GGCC-L GAACGCAGTGCTTTTCGAGG (SEQ ID NO: 152) GGCC-R ACCCTTGTTGGCCACCTCCC(SEQ ID NO: 153) GGNCC-L GGTGACAACTACTATCTGGT (SEQ ID NO: 154) GGNCC-RCTCACACCCTGCTCCGTGTA (SEQ ID NO: 155) GGNNCC-L GGGTGACAACTACTATCTGG (SEQID NO: 156) GGNNCC-R CTCACACCCTGCTCCGTGTA (SEQ ID NO: 157) GGNNNCC-LCGAGAACGCAGTGCTTTTCG (SEQ ID NO: 158) GGNNNCC-R ACCCTTGTTGGCCACCTCCC(mouse) PPP1R3C (PTG) (SEQ ID NO: 159) GGCC-L ATGAGCCAAGCAAATCCTCA (SEQID NO: 160) GGCC-R TTCCGTCATGCCCGTGGACA (SEQ ID NO: 161) GGNCC-LCTTCGTTGAAAACCATTGTA (SEQ ID NO: 162) GGNCC-R CCACCTCTGAAGAGTTTCCT (SEQID NO: 163) GGNNCC-L CTTCGTTGAAAACCATTGTA (SEQ ID NO: 164) GGNNCC-RACCACCTCTGAAGAGTTTCC (SEQ ID NO: 165) GGNNNCC-L CTTCCACTCACTCTGCGATT(SEQ ID NO: 166) GGNNNCC-R ACCATGTCTCAGTGTCAAGC PCSK9 (mouse) (SEQ IDNO: 167) GGCC-L GGCGGCAACAGCGGCAACAG (SEQ ID NO: 168) GGCC-RACTGCTCTGCGTGGCTGCGG (SEQ ID NO: 169) GGNNCC-L CCGCAGCCACGCAGAGCAGT (SEQID NO: 170) GGNNCC-R GCACCTCTCCTCGCCCCGAT

Alternative Strategies for Improving Stability of Guide RNA andIncreasing Specificity

1. Nucleotides in the 5′ of the guide RNA may be linked via thiolesterlinkages rather than phosphoester linkage like in natural RNA.Thiolester linkage may prevent the guide RNA from being digested byendogenous RNA degradation machinery.

2. Nucleotides in the guide sequence (5′ 20 bp) of the guide RNA can usebridged nucleic acids (BNA) as the bases to improve the bindingspecificity.

Example 25 CRISPR-Cas for Rapid, Multiplex Genome Editing

Aspects of the invention relate to protocols and methods by whichefficiency and specificity of gene modification may be tested within 3-4days after target design, and modified clonal cell lines may be derivedwithin 2-3 weeks.

Programmable nucleases are powerful technologies for mediating genomealteration with high precision. The RNA-guided Cas9 nuclease from themicrobial CRISPR adaptive immune system can be used to facilitateefficient genome editing in eukaryotic cells by simply specifying a20-nt targeting sequence in its guide RNA. Applicants describe a set ofprotocols for applying Cas9 to facilitate efficient genome editing inmammalian cells and generate cell lines for downstream functionalstudies. Beginning with target design, efficient and specific genemodification can be achieved within 3-4 days, and modified clonal celllines can be derived within 2-3 weeks.

The ability to engineer biological systems and organisms holds enormouspotential for applications across basic science, medicine, andbiotechnology. Programmable sequence-specific endonucleases thatfacilitate precise editing of endogenous genomic loci are now enablingsystematic interrogation of genetic elements and causal geneticvariations in a broad range of species, including those that have notbeen genetically tractable previously. A number of genome editingtechnologies have emerged in recent years, including zinc fingernucleases (ZFNs), transcription activator-like effector nucleases(TALENs), and the RNA-guided CRISPR-Cas nuclease system. The first twotechnologies use a common strategy of tethering endonuclease catalyticdomains to modular DNA-binding proteins for inducing targeted DNA doublestranded breaks (DSB) at specific genomic loci. By contrast, Cas9 is anuclease guided by small RNAs through Watson-Crick base-pairing withtarget DNA, presenting a system that is easy to design, efficient, andwell-suited for high-throughput and multiplexed gene editing for avariety of cell types and organisms. Here Applicants describe a set ofprotocols for applying the recently developed Cas9 nuclease tofacilitate efficient genome editing in mammalian cells and generate celllines for downstream functional studies.

Like ZFNs and TALENs, Cas9 promotes genome editing by stimulating DSB atthe target genomic loci. Upon cleavage by Cas9, the target locusundergoes one of two major pathways for DNA damage repair, theerror-prone non-homologous end joining (NHEJ) or the high-fidelityhomology directed repair (HDR) pathway. Both pathways may be utilized toachieve the desired editing outcome.

NHEJ: In the absence of a repair template, the NHEJ process re-ligatesDSBs, which may leave a scar in the form of indel mutations. Thisprocess can be harnessed to achieve gene knockouts, as indels occurringwithin a coding exon may lead to frameshift mutations and a prematurestop codon. Multiple DSBs may also be exploited to mediate largerdeletions in the genome.

HDR: Homology directed repair is an alternate major DNA repair pathwayto NHEJ. Although HDR typically occurs at lower frequencies than NHEJ,it may be harnessed to generate precise, defined modifications at atarget locus in the presence of an exogenously introduced repairtemplate. The repair template may be either in the form of doublestranded DNA, designed similarly to conventional DNA targetingconstructs with homology arms flanking the insertion sequence, orsingle-stranded DNA oligonucleotides (ssODNs). The latter provides aneffective and simple method for making small edits in the genome, suchas the introduction of single nucleotide mutations for probing causalgenetic variations. Unlike NHEJ, HDR is generally active only individing cells and its efficiency varies depending on the cell type andstate.

Overview of CRISPR: The CRISPR-Cas system, by contrast, is at minimum atwo-component system consisting of the Cas9 nuclease and a short guideRNA. Re-targeting of Cas9 to different loci or simultaneous editing ofmultiple genes simply requires cloning a different 20-bpoligonucleotide. Although specificity of the Cas9 nuclease has yet to bethoroughly elucidated, the simple Watson-Crick base-pairing of theCRISPR-Cas system is likely more predictable than that of ZFN or TALENdomains.

The type II CRISPR-Cas (clustered regularly interspaced shortpalindromic repeats) is a bacterial adaptive immune system that usesCas9, to cleave foreign genetic elements. Cas9 is guided by a pair ofnon-coding RNAs, a variable crRNA and a required auxiliary tracrRNA. ThecrRNA contains a 20-nt guide sequence determines specificity by locatingthe target DNA via Watson-Crick base-pairing. In the native bacterialsystem, multiple crRNAs are co-transcribed to direct Cas9 againstvarious targets. In the CRISPR-Cas system derived from Streptococcuspyogenes, the target DNA must immediately precede a 5′-NGG/NRGprotospacer adjacent motif (PAM), which can vary for other CRISPRsystems.

CRISPR-Cas is reconstituted in mammalian cells through the heterologousexpression of human codon-optimized Cas9 and the requisite RNAcomponents. Furthermore, the crRNA and tracrRNA can be fused to create achimeric, synthetic guide RNA (sgRNA). Cas9 can thus be re-directedtoward any target of interest by altering the 20-nt guide sequencewithin the sgRNA.

Given its ease of implementation and multiplex capability, Cas9 has beenused to generate engineered eukaryotic cells carrying specific mutationsvia both NHEJ and HDR. In addition, direct injection of sgRNA and mRNAencoding Cas9 into embryos has enabled the rapid generation oftransgenic mice with multiple modified alleles; these results holdpromise for editing organisms that are otherwise geneticallyintractable.

A mutant Cas9 carrying a disruption in one of its catalytic domains hasbeen engineered to nick rather than cleave DNA, allowing forsingle-stranded breaks and preferential repair through HDR, potentiallyameliorating unwanted indel mutations from off-target DSBs.Additionally, a Cas9 mutant with both DNA-cleaving catalytic residuesmutated has been adapted to enable transcriptional regulation in E.coli, demonstrating the potential of functionalizing Cas9 for diverseapplications. Certain aspects of the invention relate to theconstruction and application of Cas9 for multiplexed editing of humancells.

Applicants have provided a human codon-optimized, nuclear localizationsequence-flanked Cas9 to facilitate eukaryotic gene editing. Applicantsdescribe considerations for designing the 20-nt guide sequence,protocols for rapid construction and functional validation of sgRNAs,and finally use of the Cas9 nuclease to mediate both NHEJ- and HDR-basedgenome modifications in human embryonic kidney (HEK-293FT) and humanstem cell (HUES9) lines. This protocol can likewise be applied to othercell types and organisms.

Target selection for sgRNA: There are two main considerations in theselection of the 20-nt guide sequence for gene targeting: 1) the targetsequence should precede the 5′-NGG PAM for S. pyogenes Cas9, and 2)guide sequences should be chosen to minimize off-target activity.Applicants provided an online Cas9 targeting design tool that takes aninput sequence of interest and identifies suitable target sites. Toexperimentally assess off-target modifications for each sgRNA,Applicants also provide computationally predicted off-target sites foreach intended target, ranked according to Applicants' quantitativespecificity analysis on the effects of base-pairing mismatch identity,position, and distribution.

The detailed information on computationally predicted off-target sitesis as follows:

Considerations for Off-target Cleavage Activities: Similar to othernucleases, Cas9 can cleave off-target DNA targets in the genome atreduced frequencies. The extent to which a given guide sequence exhibitoff-target activity depends on a combination of factors including enzymeconcentration, thermodynamics of the specific guide sequence employed,and the abundance of similar sequences in the target genome. For routineapplication of Cas9, it is important to consider ways to minimize thedegree of off-target cleavage and also to be able to detect the presenceof off-target cleavage.

Minimizing off-target activity: For application in cell lines,Applicants recommend following two steps to reduce the degree ofoff-target genome modification. First, using our online CRISPR targetselection tool, it is possible to computationally assess the likelihoodof a given guide sequence to have off-target sites. These analyses areperformed through an exhaustive search in the genome for off-targetsequences that are similar sequences as the guide sequence.Comprehensive experimental investigation of the effect of mismatchingbases between the sgRNA and its target DNA revealed that mismatchtolerance is 1) position dependent—the 8-14 bp on the 3′ end of theguide sequence are less tolerant of mismatches than the 5′ bases, 2)quantity dependent—in general more than 3 mismatches are not tolerated,3) guide sequence dependent—some guide sequences are less tolerant ofmismatches than others, and 4) concentration dependent—off-targetcleavage is highly sensitive to the amount of transfected DNA. TheApplicants' target site analysis web tool (available at the websitegenome-engineering.org/tools) integrates these criteria to providepredictions for likely off-target sites in the target genome. Second,Applicants recommend titrating the amount of Cas9 and sgRNA expressionplasmid to minimize off-target activity.

Detection of off-target activities: Using Applicants' CRISPR targetingweb tool, it is possible to generate a list of most likely off-targetsites as well as primers performing SURVEYOR or sequencing analysis ofthose sites. For isogenic clones generated using Cas9, Applicantsstrongly recommend sequencing these candidate off-target sites to checkfor any undesired mutations. It is worth noting that there may be offtarget modifications in sites that are not included in the predictedcandidate list and full genome sequence should be performed tocompletely verify the absence of off-target sites. Furthermore, inmultiplex assays where several DSBs are induced within the same genome,there may be low rates of translocation events and can be evaluatedusing a variety of techniques such as deep sequencing.

The online tool provides the sequences for all oligos and primersnecessary for 1) preparing the sgRNA constructs, 2) assaying targetmodification efficiency, and 3) assessing cleavage at potentialoff-target sites. It is worth noting that because the U6 RNA polymeraseIII promoter used to express the sgRNA prefers a guanine (G) nucleotideas the first base of its transcript, an extra G is appended at the 5′ ofthe sgRNA where the 20-nt guide sequence does not begin with G.

Approaches for sgRNA construction and delivery: Depending on the desiredapplication, sgRNAs may be delivered as either 1) PCR ampliconscontaining an expression cassette or 2) sgRNA-expressing plasmids.PCR-based sgRNA delivery appends the custom sgRNA sequence onto thereverse PCR primer used to amplify a U6 promoter template. The resultingamplicon may be co-transfected with a plasmid containing Cas9 (PX165).This method is optimal for rapid screening of multiple candidate sgRNAs,as cell transfections for functional testing can be performed mere hoursafter obtaining the sgRNA-encoding primers. Because this simple methodobviates the need for plasmid-based cloning and sequence verification,it is well suited for testing or co-transfecting a large number ofsgRNAs for generating large knockout libraries or other scale-sensitiveapplications. Note that the sgRNA-encoding primers are over 100-bp,compared to the ˜20-bp oligos required for plasmid-based sgRNA delivery.

Construction of an expression plasmid for sgRNA is also simple andrapid, involving a single cloning step with a pair of partiallycomplementary oligonucleotides. After annealing the oligo pairs, theresulting guide sequences may be inserted into a plasmid bearing bothCas9 and an invariant scaffold bearing the remainder of the sgRNAsequence (PX330). The transfection plasmids may also be modified toenable virus production for in vivo delivery.

In addition to PCR and plasmid-based delivery methods, both Cas9 andsgRNA can be introduced into cells as RNA.

Design of repair template: Traditionally, targeted DNA modificationshave required use of plasmid-based donor repair templates that containhomology arms flanking the site of alteration. The homology arms on eachside can vary in length, but are typically longer than 500 bp. Thismethod can be used to generate large modifications, including insertionof reporter genes such as fluorescent proteins or antibiotic resistancemarkers. The design and construction of targeting plasmids has beendescribed elsewhere.

More recently, single-stranded DNA oligonucleotides (ssODNs) have beenused in place of targeting plasmids for short modifications within adefined locus without cloning. To achieve high HDR efficiencies, ssODNscontain flanking sequences of at least 40 bp on each side that arehomologous to the target region, and can be oriented in either the senseor antisense direction relative to the target locus.

Functional Testing

SURVEYOR nuclease assay: Applicants detected indel mutations either bythe SURVEYOR nuclease assay (or PCR amplicon sequencing. Applicantsonline CRISPR target design tool provides recommended primers for bothapproaches. However, SURVEYOR or sequencing primers may also be designedmanually to amplify the region of interest from genomic DNA and to avoidnon-specific amplicons using NCBI Primer-BLAST. SURVEYOR primers shouldbe designed to amplify 300-400 bp (for a 600-800 bp total amplicon) oneither side of the Cas9 target for allowing clear visualization ofcleavage bands by gel electrophoresis. To prevent excessive primer dimerformation, SURVEYOR primers should be designed to be typically under25-nt long with melting temperatures of ˜60° C. Applicants recommendtesting each pair of candidate primers for specific PCR amplicons aswell as for the absence of non-specific cleavage during the SURVEYORnuclease digestion process.

Plasmid- or ssODN-mediated HDR: HDR can be detected viaPCR-amplification and sequencing of the modified region. PCR primers forthis purpose should anneal outside the region spanned by the homologyarms to avoid false detection of residual repair template (HDR Fwd andRev, FIG. 12). For ssODN-mediated HDR, SURVEYOR PCR primers can be used.

Detection of indels or HDR by sequencing: Applicants detected targetedgenome modifications by either Sanger or next-generation deep sequencing(NGS). For the former, genomic DNA from modified region can be amplifiedusing either SURVEYOR or HDR primers. Amplicons should be subcloned intoa plasmid such as pUC19 for transformation; individual colonies can besequenced to reveal clonal genotype.

Applicants designed next-generation sequencing (NGS) primers for shorteramplicons, typically in the 100-200 bp size range. For detecting NHEJmutations, it is important to design primers with at least 10-20 bpbetween the priming regions and the Cas9 target site to allow detectionof longer indels. Applicants provide guidelines for a two-step PCRmethod to attach barcoded adapters for multiplex deep sequencing.Applicants recommend the Illumina platform, due to its generally lowlevels of false positive indels. Off-target analysis (describedpreviously) can then be performed through read alignment programs suchas ClustalW, Geneious, or simple sequence analysis scripts.

Materials and Reagents

sgRNA Preparation:

UltraPure DNaseRNase-free distilled water (Life Technologies, cat. no.10977-023)

Herculase II fusion polymerase (Agilent Technologies, cat. no. 600679)

CRITICAL. Standard Taq polymerase, which lacks 3′-5′ exonucleaseproofreading activity, has lower fidelity and can lead to amplificationerrors. Herculase II is a high-fidelity polymerase (equivalent fidelityto Pfu) that produces high yields of PCR product with minimaloptimization. Other high-fidelity polymerases may be substituted.

Herculase II reaction buffer (5×; Agilent Technologies, included withpolymerase)

dNTP solution mix (25 mM each; Enzymatics, cat. no. N205L)

MgCl2 (25 mM; ThermoScientific, cat. no. R0971)

QIAquick gel extraction kit (Qiagen, cat. no. 28704)

QIAprep spin miniprep kit (Qiagen, cat. no. 27106)

UltraPure TBE buffer (10×; Life Technologies, cat. no. 15581-028)

SeaKem LE agarose (Lonza, cat. no. 50004)

SYBR Safe DNA stain (10,000×; Life Technologies, cat. no. S33102)

1-kb Plus DNA ladder (Life Technologies, cat. no. 10787-018)

TrackIt CyanOrange loading buffer (Life Technologies, cat. no.10482-028)

FastDigest BbsI (BpiI) (Fermentas/ThermoScientific, cat. no. FD1014)

Fermentas Tango Buffer (Fermentas/ThermoScientific, cat. no. BY5)

DL-dithiothreitol (DTT; Fermentas/ThermoScientific, cat. no. R0862)

T7 DNA ligase (Enzymatics, cat. no. L602L)

Critical:Do not substitute the more commonly used T4 ligase. T7 ligasehas 1,000-fold higher activity on the sticky ends than on the blunt endsand higher overall activity than commercially available concentrated T4ligases.

T7 2× Rapid Ligation Buffer (included with T7 DNA ligase, Enzymatics,cat. no. L602L)

T4 Polynucleotide Kinase (New England Biolabs, cat. no MO201S)

T4 DNA Ligase Reaction Buffer (10×; New England Biolabs, cat. no B0202S)

Adenosine 5′-triphosphate (10 mM; New England Biolabs, cat. no. P0756S)

PlasmidSafe ATP-dependent DNase (Epicentre, cat. no. E3101K)

One Shot Stb13 chemically competent Escherichia coli (E. coli) (LifeTechnologies, cat. no. C7373-03)

SOC medium (New England Biolabs, cat. no. B9020S)

LB medium (Sigma, cat. no. L3022)

LB agar medium (Sigma, cat. no. L2897)

Ampicillin, sterile filtered (100 mg ml-1; Sigma, cat. no. A5354)

Mammalian Cell Culture:

HEK293FT cells (Life Technologies, cat. no. R700-07)

Dulbecco's minimum Eagle's medium (DMEM, 1×, high glucose; LifeTechnologies, cat. no. 10313-039)

Dulbecco's minimum Eagle's medium (DMEM, 1×, high glucose, no phenolred; Life Technologies, cat. no. 31053-028)

Dulbecco's phosphate-buffered saline (DPBS, 1×; Life Technologies, cat.no. 14190-250)

Fetal bovine serum, qualified and heat inactivated (Life Technologies,cat. no. 10438-034)

Opti-MEM I reduced-serum medium (FBS; Life Technologies, cat. no.11058-021)

Penicillin-streptomycin (100×; Life Technologies, cat. no. 15140-163)

TrypLE™ Express (1×, no Phenol Red; Life Technologies, cat. no.12604-013)

Lipofectamine 2000 transfection reagent (Life Technologies, cat. no.11668027)

Amaxa SF Cell Line 4D-Nucleofector® X Kit S (32 RCT; Lonza, cat. noV4XC-2032)

HUES 9 cell line (HARVARD STEM CELL SCIENCE)

Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix (LifeTechnologies, cat. no. A1413201)

mTeSR1 medium (Stemcell Technologies, cat. no. 05850)

Accutase cell detachment solution (Stemcell Technologies, cat. no.07920)

ROCK Inhibitor (Y-27632; Millipore, cat. no. SCM075)

Amaxa P3 Primary Cell 4D-Nucleofector® X Kit S (32 RCT; Lonza cat. no.V4XP-3032)

Genotyping Analysis:

QuickExtract DNA extraction solution (Epicentre, cat. no. QE09050)

PCR primers for SURVEYOR, RFLP analysis, or sequencing (see Primertable)

Herculase II fusion polymerase (Agilent Technologies, cat. no. 600679)

CRITICAL. As Surveyor assay is sensitive to single-base mismatches, itis particularly important to use a high-fidelity polymerase. Otherhigh-fidelity polymerases may be substituted.

Herculase II reaction buffer (5×; Agilent Technologies, included withpolymerase)

dNTP solution mix (25 mM each; Enzymatics, cat. no. N205L)

QIAquick gel extraction kit (Qiagen, cat. no. 28704)

Taq Buffer (10×; Genscript, cat. no. B0005)

SURVEYOR mutation detection kit for standard gel electrophoresis(Transgenomic, cat. no. 706025)

UltraPure TBE buffer (10×; Life Technologies, cat. no. 15581-028)

SeaKem LE agarose (Lonza, cat. no. 50004)

4-20% TBE Gels 1.0 mm, 15 Well (Life Technologies, cat. no. EC62255BOX)

Novex® Hi-Density TBE Sample Buffer (5×; Life Technologies, cat. no.LC6678)

SYBR Gold Nucleic Acid Gel Stain (10,000×; Life Technologies, cat. no.S-11494)

1-kb Plus DNA ladder (Life Technologies, cat. no. 10787-018)

TrackIt CyanOrange loading buffer (Life Technologies, cat. no.10482-028)

FastDigest HindIII (Fermentas/ThermoScientific, cat. no. FD0504)

Equipment

Filtered sterile pipette tips (Corning)

Standard 1.5 ml microcentrifuge tubes (Eppendorf, cat. no. 0030 125.150)

Axygen 96-well PCR plates (VWR, cat. no. PCR-96M2-HSC)

Axygen 8-Strip PCR tubes (Fischer Scientific, cat. no. 14-222-250)

Falcon tubes, polypropylene., 15 ml (BD Falcon, cat. no. 352097)

Falcon tubes, polypropylene, 50 m (BD Falcon, cat. no. 352070)

Round-bottom Tube with cell strainer cap, 5 ml (BD Falcon, cat. no.352235)

Petri dishes (60 mm×15 mm; BD Biosciences, cat. no. 351007)

Tissue culture plate (24 well; BD Falcon, cat. no. 353047)

Tissue culture plate (96 well, flat bottom; BD Falcon, cat. no. 353075)

Tissue culture dish 100 mm; BD Falcon, 353003)

96-well thermocycler with programmable temperature steppingfunctionality (Applied Biosystems Veriti, cat. no. 4375786).

Desktop microcentrifuges 5424, 5804 (Eppendorf)

Gel electrophoresis system (PowerPac basic power supply, Bio-Rad, cat.no. 164-5050, and Sub-Cell GT System gel tray, Bio-Rad, cat. no.170-4401)

Novex XCe11 SureLock Mini-Cell (Life Technologies, cat. no. EI0001)

Digital gel imaging system (GelDoc EZ, Bio-Rad, cat. no. 170-8270, andblue sample tray, Bio-Rad, cat. no. 170-8273)

Blue light transilluminator and orange filter goggles (SafeImager 2.0;Invitrogen, cat. no. G6600)

Gel quantification software (Bio-Rad, ImageLab, included with GelDoc EZ,or open-source ImageJ from the National Institutes of Health, availableat the website rsbweb.nih.gov/ij/) UV spectrophotometer (NanoDrop 2000c,Thermo Scientific)

Reagent Setup

Tris-borate EDTA (TBE) electrophoresis solution Dilute TBE buffer indistilled water to 1× working solution for casting agarose gels and foruse as a buffer for gel electrophoresis. Buffer may be stored at roomtemperature (18-22° C.) for at least 1 year.

-   -   ATP, 10 mM Divide 10 mM ATP into 50-μl aliquots and store at        −20° C. for up to 1 year; avoid repeated freeze-thaw cycles.    -   DTT, 10 mM Prepare 10 mM DTT solution in distilled water and        store in 20-μl aliquots at −70° C. for up to 2 years; for each        reaction, use a new aliquot, as DTT is easily oxidized.    -   D10 culture medium For culture of HEK293FT cells, prepare D10        culture medium by supplementing DMEM with 1× GlutaMAX and 10%        (vol/vol) fetal bovine serum. As indicated in the protocol, this        medium can also be supplemented with 1× penicillin-streptomycin.        D10 medium can be made in advance and stored at 4° C. for up to        1 month.    -   mTeSR1 culture medium For culture of human embryonic stem cells,        prepare mTeSR1 medium by supplementing the 5× supplement        (included with mTeSR1 basal medium), and 100 ug/ml Normocin.

Procedure

Design of targeting components and use of the online tool • Timing 1 d

1| Input target genomic DNA sequence. Applicants provide an online Cas9targeting design tool that takes an input sequence of interest,identifies and ranks suitable target sites, and computationally predictsoff-target sites for each intended target. Alternatively, one canmanually select guide sequence by identifying the 20-bp sequencedirectly upstream of any 5′-NGG.

2| Order necessary oligos and primers as specified by the online tool.If the site is chosen manually, the oligos and primers should bedesigned.

Preparation of sgRNA expression construct

3| To generate the sgRNA expression construct, either the PCR- orplasmid-based protocol can be used.

(A) via PCR amplification • Timing 2 h

(i) Applicants prepare diluted U6 PCR template. Applicants recommendusing PX330 as a PCR template, but any U6-containing plasmid maylikewise be used as the PCR template. Applicants diluted template withddH₂O to a concentration of 10 ng/ul. Note that if a plasmid or cassettealready containing an U6-driven sgRNA is used as a template, a gelextraction needs to be performed to ensure that the product containsonly the intended sgRNA and no trace sgRNA carryover from template.

(ii) Applicants prepared diluted PCR oligos. U6-Fwd and U6-sgRNA-Revprimers are diluted to a final concentration of 10 uM in ddH₂O (add 10ul of 100 uM primer to 90 ul ddH₂O).

(iii) U6-sgRNA PCR reaction. Applicants set up the following reactionfor each U6-sgRNA-Rev primer and mastermix as needed:

Final Component: Amount (ul) concentration Herculase II PCR buffer, 5X10 1X dNTP, 100 mM (25 mM each) 0.5 1 mM U6 template (PX330) 1 0.2 ng/ulU6-Fwd primer 1 0.2 uM U6-sgRNA-Rev primer (variable) 1 0.2 uM HerculaseII Fusion polymerase 0.5 Distilled water 36 Total 50

(iv) Applicants performed PCR reaction on the reactions from step (iii)using the following cycling conditions:

Cycle number Denature Anneal Extend  1 95° C., 2 m 2-31 95° C., 20 s 60°C., 20 s 72° C., 20 s 32 72° C., 3 m

(v) After the reaction is completed, Applicants ran the product on a gelto verify successful, single-band amplification. Cast a 2% (wt/vol)agarose gel in 1×TBE buffer with 1×SYBR Safe dye. Run 5 ul of the PCRproduct in the gel at 15 V cm-1 for 20-30 min. Successful ampliconsshould yield one single 370-bp product and the template should beinvisible. It should not be necessary to gel extract the PCR amplicon.

(vi) Applicants purified the PCR product using the QIAquick PCRpurification kit according to the manufacturer's directions. Elute theDNA in 35 ul of Buffer EB or water. Purified PCR products may be storedat 4° C. or −20° C.

(B) Cloning sgRNA into Cas9-containing bicistronic expression vector •Timing 3 d

(i) Prepare the sgRNA Oligo Inserts.

Applicants resuspended the top and bottom strands of oligos for eachsgRNA design to a final concentration of 100 uM. Phosphorylate andanneal the oligo as follows:

Oligo 1 (100 uM) 1 ul Oligo 2 (100 uM) 1 ul T4 Ligation Buffer, 10X 1 ulT4 PNK 1 ul ddH₂O 6 ul Total 10 ul 

(ii) Anneal in a thermocycler using the following parameters:

-   -   37° C. for 30 m    -   95° C. for 5 m    -   Ramp down to 25° C. at 5° C. per m

(iii) Applicants diluted phosphorylated and annealed oligos 1:200 by add1 ul of oligo to 199 ul room temperature ddH₂O.

(iv) Clone sgRNA Oligo into PX330.

Applicants set up Golden Gate reaction for each sgRNA. Applicantsrecommend also setting up a no-insert, PX330 only negative control.

PX330 (100 ng) x ul Diluted oligo duplex from step (iii) 2 ul TangoBuffer, 10X 2 ul DTT, 10 mM 1 ul ATP, 10 mM 1 ul FastDigest BbsI 1 ul T7Ligase 0.5 ul ddH₂O x ul Total 20 ul

(v) Incubate the Golden Gate reaction for a total of 1 h:

Cycle number Condition 1-6 37° C. for 5 m, 21° C. for 5 m

(vi) Applicants treated Golden Gate reaction with PlasmidSafeexonuclease to digest any residual linearized DNA. This step is optionalbut highly recommended.

Golden Gate reaction from step 4 11 ul 10X PlasmidSafe Buffer 1.5 ulATP, 10 mM 1.5 ul PlasmidSafe exonuclease 1 ul Total 15 ul

(vii) Applicants incubated the PlasmidSafe reaction at 37° C. for 30min, followed by inactivation at 70° C. for 30 min. Pause point: aftercompletion, the reaction may be frozen and continued later. The circularDNA should be stable for at least 1 week.

(viii) Transformation.

Applicants transformed the PlasmidSafe-treated plasmid into a competentE. coli strain, according to the protocol supplied with the cells.Applicants recommend Stb13 for quick transformation. Briefly, Applicantsadded 5 ul of the product from step (vii) into 20 ul of ice-coldchemically competent Stb13 cells. This is then incubated on ice for 10m, heat shocked at 42° C. for 30 s, returned immediately to ice for 2 m,100 ul of SOC medium is added, and this is plated onto an LB platecontaining 100 ug/ml ampicillin with incubation overnight at 37° C.

(ix) Day 2: Applicants inspected plates for colony growth. Typically,there are no colonies on the negative control plates (ligation ofBbsI-digested PX330 only, no annealed sgRNA oligo), and tens to hundredsof colonies on the PX330-sgRNA cloning plates.

(x) From each plate, Applicants picked 2-3 colonies to check correctinsertion of sgRNA. Applicants used a sterile pipette tip to inoculate asingle colony into a 3 ml culture of LB medium with 100 ug/mlampicillin. Incubate and shake at 37° C. overnight.

(xi) Day 3: Applicants isolated plasmid DNA from overnight culturesusing a QiAprep Spin miniprep kit according to the manufacturer'sinstructions.

(xii) Sequence Validate CRISPR Plasmid.

Applicants verified the sequence of each colony by sequencing from theU6 promoter using the U6-Fwd primer. Optional: sequence the Cas9 geneusing primers listed in the following Primer table.

Primer Sequence (5′ to 3′) Purpose U6-For GAGGGCCTATTTCCCATGATTCC (SEQID NO: 171) Amplify U6- sgRNA U6-RevAAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGAT Amplify U6-AACGGACTAGCCTTATTTTAACTTGCTATTTCTAG sgRNA; N isCTCTAAAACNNNNNNNNNNNNNNNNNNNCCGGTGTTTC reverse GTCCTTTCCACAAG (SEQ IDNO: 172) complement of target sgRNA- CACCGNNNNNNNNNNNNNNNNNNN (SEQ IDNO: 173) Clone sgRNA top into PX330 sgRNA- AAACNNNNNNNNNNNNNNNNNNNC (SEQID NO: 174) Clone sgRNA bottom into PX330 U6-EMX1-AAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGAT Amplify U6- RevAACGGACTAGCCTTATTTTAACTTGCTATTTCTAG EMX1 sgRNACTCTAAAACCCCTAGTCATTGGAGGTGACCGGTGTTTCG TCCTTTCCACAAG (SEQ ID NO: 175)EMX1-top CACCGTCACCTCCAATGACTAGGG (SEQ ID NO: 176) Clone EMX1 sgRNA intoPX330 EMX1- AAACCCCTAGTCATTGGAGGTGAC (SEQ ID NO: 177) Clone EMX1 bottomsgRNA into PX330 ssODN- CAGAAGAAGAAGGGCTCCCATCACATCAACCGGTGGCG EMX1 HDRsense CATTGCCACGAAGCAGGCCAATGGGGAGGACATC (sense;GATGTCACCTCCAATGACAAGCTTGCTAGCGGTGGGCAA insertionCCACAAACCCACGAGGGCAGAGTGCTGCTTGCTG underlined)CTGGCCAGGCCCCTGCGTGGGCCCAAGCTGGACTCTGGC CACTCCCT (SEQ ID NO: 178) ssODN-AGGGAGTGGCCAGAGTCCAGCTTGGGCCCACGCAGGGG EMX1 HDR antisenseCCTGGCCAGCAGCAAGCAGCACTCTGCCCTCGTG (antisense;GGTTTGTGGTTGCCCACCGCTAGCAAGCTTGTCATTGGA insertionGGTGACATCGATGTCCTCCCCATTGGCCTGCTTCG underlined)TGGCAATGCGCCACCGGTTGATGTGATGGGAGCCCTTCT TCTTCTG (SEQ ID NO: 179) EMX1-CCATCCCCTTCTGTGAATGT (SEQ ID NO: 180) EMX1 SURV-F SURVEYOR assay PCR,sequencing EMX1- GGAGATTGGAGACACGGAGA (SEQ ID NO: 181) EMX1 SURV-RSURVEYOR assay PCR, sequencing EMX1- GGCTCCCTGGGTTCAAAGTA (SEQ ID NO:182) EMX1 RFLP HDR-F analysis PCR, sequencing EMX1-AGAGGGGTCTGGATGTCGTAA (SEQ ID NO: 183) EMX1 RFLP HDR-R analysis PCR,sequencing pUC19-F CGCCAGGGTTTTCCCAGTCACGAC (SEQ ID NO: 184) pUC19multiple cloning site F primer, for Sanger sequencing

Applicants referenced the sequencing results against the PX330 cloningvector sequence to check that the 20 bp guide sequence was insertedbetween the U6 promoter and the remainder of the sgRNA scaffold. Detailsand sequence of the PX330 map in GenBank vector map format (*.gb file)can be found at the website crispr.genome-engineering.org.

(Optional) Design of ssODN Template • Timing 3 d Planning Ahead

3| Design and Order ssODN.

Either the sense or antisense ssODN can be purchased directly fromsupplier. Applicants recommend designing homology arms of at least 40 bpon either side and 90 bp for optimal HDR efficiency. In Applicants'experience, antisense oligos have slightly higher modificationefficiencies.

4| Applicants resuspended and diluted ssODN ultramers to a finalconcentration of 10 uM. Do not combine or anneal the sense and antisensessODNs. Store at −20° C.

5| Note for HDR applications, Applicants recommend cloning sgRNA intothe PX330 plasmid.

Functional Validation of sgRNAs: Cell Culture and Transfections • Timing3-4 d

The CRISPR-Cas system has been used in a number of mammalian cell lines.Conditions may vary for each cell line. The protocols below detailstransfection conditions for HEK239FT cells. Note for ssODN-mediated HDRtransfections, the Amaxa SF Cell Line Nucleofector Kit is used foroptimal delivery of ssODNs. This is described in the next section.

7| HEK293FT Maintenance.

Cells are maintained according to the manufacturer's recommendations.Briefly, Applicants cultured cells in D10 medium (GlutaMax DMEMsupplemented with 10% Fetal Bovine Serum), at 37° C. and 5% CO2.

8| To passage, Applicants removed medium and rinsed once by gentlyadding DPBS to side of vessel, so as not to dislodge cells. Applicantsadded 2 ml of TrypLE to a T75 flask and incubated for 5 m at 37° C. 10ml of warm D10 medium is added to inactivate and transferred to a 50 mlFalcon tube. Applicants dissociated cells by triturating gently, andre-seeded new flasks as necessary. Applicants typically passage cellsevery 2-3 d at a split ratio of 1:4 or 1:8, never allowing cells toreach more than 70% confluency. Cell lines are restarted upon reachingpassage number 15.

9| Prepare Cells for Transfection.

Applicants plated well-dissociated cells onto 24-well plates in D10medium without antibiotics 16-24 h before transfection at a seedingdensity of 1.3×10⁵ cells per well and a seeding volume of 500 ul. Scaleup or down according to the manufacturer's manual as needed. It issuggested to not plate more cells than recommended density as doing somay reduce transfection efficiency.

10| On the day of transfection, cells are optimal at 70-90% confluency.Cells may be transfected with Lipofectamine 2000 or Amaxa SF Cell LineNucleofector Kit according to the manufacturers' protocols.

(A) For sgRNAs cloned into PX330, Applicants transfected 500 ng ofsequence-verified CRISPR plasmid; if transfecting more than one plasmid,mix at equimolar ratio and no more than 500 ng total.

(B) For sgRNA amplified by PCR, Applicants mixed the following:

PX165 (Cas9 only) 200 ng sgRNA amplicon (each)  40 ng pUC19 fill uptotal DNA to 500 ng

Applicants recommend transfecting in technical triplicates for reliablequantification and including transfection controls (e.g. GFP plasmid) tomonitor transfection efficiency. In addition, PX330 cloning plasmidand/or sgRNA amplicon may be transfected alone as a negative control fordownstream functional assays.

11| Applicants added Lipofectamine complex to cells gently as HEK293FTcells may detach easily from plate easily and result in lowertransfection efficiency.

12| Applicants checked cells 24 h after transfection for efficiency byestimating the fraction of fluorescent cells in the control (e.g., GFP)transfection using a fluorescence microscope. Typically cells are morethan 70% transfected.

13| Applicants supplemented the culture medium with an additional 500 ulof warm D10 medium. Add D10 very slowly to the side of the well and donot use cold medium, as cells can detach easily.

14| Cells are incubated for a total of 48-72 h post-transfection beforeharvested for indel analysis. Indel efficiency does not increasenoticeably after 48 h.

(Optional) Co-Transfection of CRISPR Plasmids and ssODNs or TargetingPlasmids for HR • Timing 3-4 d

15| Linearize Targeting Plasmid.

Targeting vector is linearized if possible by cutting once at arestriction site in the vector backbone near one of the homology arms orat the distal end of either homology arm.

16| Applicants ran a small amount of the linearized plasmid alongsideuncut plasmid on a 0.8-1% agarose gel to check successful linearization.Linearized plasmid should run above the supercoiled plasmid.

17| Applicants purified linearized plasmid with the QIAQuick PCRPurification kit.

18| Prepare cells for transfection. Applicants cultured HEK293FT in T75or T225 flasks. Sufficient cell count before day of transfection isplanned for. For the Amaxa strip-cuvette format, 2×10⁶ cells are usedper transfection.

19| Prepare plates for transfection. Applicants added 1 ml of warm D10medium into each well of a 12 well plate. Plates are placed into theincubator to keep medium warm.

20|Nucleofection. Applicants transfected HEK293FT cells according to theAmaxa SF Cell Line Nucleofector 4D Kit manufacturer's instructions,adapted in the steps below.

-   -   a. For ssODN and CRISPR cotransfection, pre-mix the following        DNA in PCR tubes:

pCRISPR plasmid (Cas9 + sgRNA) 500 ng ssODN template (10 uM) 1 ul

-   -   b. For HDR targeting plasmid and CRISPR cotransfection, pre-mix        the following DNA in PCR tubes:

CRISPR plasmid (Cas9 + sgRNA) 500 ng Linearized targeting plasmid 500 ng

For transfection controls, see previous section. In addition, Applicantsrecommend transfecting ssODN or targeting plasmid alone as a negativecontrol.

21| Dissociate to single cells. Applicants removed medium and rinsedonce gently with DPBS, taking care not to dislodge cells. 2 ml of TrypLEis added to a T75 flask and incubated for 5 m at 37° C. 10 ml of warmD10 medium is added to inactivate and triturated gently in a 50 mlFalcon tube. It is recommended that cells are triturated gently anddissociated to single cells. Large clumps will reduce transfectionefficiency. Applicants took a 10 ul aliquot from the suspension anddiluted into 90 ul of D10 medium for counting. Applicants counted cellsand calculated the number of cells and volume of suspension needed fortransfection. Applicants typically transfected 2×10⁵ cells per conditionusing the Amaxa Nucleocuvette strips, and recommend calculating for 20%more cells than required to adjust for volume loss in subsequentpipetting steps. The volume needed is transferred into a new Falcontube.

23| Applicants spun down the new tube at 200×g for 5 m.

Applicants prepared the transfection solution by mixing the SF solutionand S1 supplement as recommended by Amaxa. For Amaxa strip-cuvettes, atotal of 20 ul of supplemented SF solution is needed per transfection.Likewise, Applicants recommend calculating for 20% more volume thanrequired.

25| Applicants removed medium completely from pelleted cells from step23 and gently resuspended in appropriate volume (20 ul per 2×10⁵ cells)of 51-supplemented SF solution. Do not leave cells in SF solution forextended period of time.

26| 20 ul of resuspended cells is pipetted into each DNA pre-mix fromstep 20. Pipette gently to mix and transfer to Nucleocuvette stripchamber. This is repeated for each transfection condition.

Electroporate cells using the Nucleofector 4D program recommended byAmaxa, CM-130.

28| Applicants gently and slowly pipetted 100 ul of warm D10 medium intoeach Nucleocuvette strip chamber, and transferred all volume into thepre-warmed plate from step 19. CRITICAL. Cells are very fragile at thisstage and harsh pipetting can cause cell death. Incubate for 24 h. Atthis point, transfection efficiency can be estimated from fraction offluorescent cells in positive transfection control. Nucleofectiontypically results in greater than 70-80% transfection efficiency.Applicants slowly added 1 ml warm D10 medium to each well withoutdislodging the cells. Incubate cells for a total of 72 h.

Human Embryonic Stem Cell (HUES 9) Culture and Transfection • Timing 3-4d

Maintaining hESC (HUES9) line. Applicants routinely maintain HUES9 cellline in feeder-free conditions with mTesR1 medium. Applicants preparedmTeSR1 medium by adding the 5× supplement included with basal medium and100 ug/ml Normocin. Applicants prepared a 10 ml aliquot of mTeSR1 mediumsupplemented further with 10 uM Rock Inhibitor. Coat tissue cultureplate. Dilute cold GelTrex 1:100 in cold DMEM and coat the entiresurface of a 100 mm tissue culture plate.

Place plate in incubator for at least 30 m at 37° C. Thaw out a vial ofcells at 37° C. in a 15 ml Falcon tube, add 5 ml of mTeSR1 medium, andpellet at 200×g for 5 m. Aspirate off GelTrex coating and seed ˜1×106cells with 10 ml mTeSR1 medium containing Rock Inhibitor. Change tonormal mTeSR1 medium 24 h after transfection and re-feed daily.Passaging cells. Re-feed cells with fresh mTeSR1 medium daily andpassage before reaching 70% confluency. Aspirate off mTeSR1 medium andwash cells once with DPBS. Dissociate cells by adding 2 ml Accutase andincubating at 37° C. for 3-5 m. Add 10 ml mTeSR1 medium to detachedcells, transfer to 15 ml Falcon tube and resuspend gently. Re-plate ontoGelTrex-coated plates in mTeSR1 medium with 10 uM Rock Inhibitor. Changeto normal mTeSR1 medium 24 h after plating.

Transfection. Applicants recommend culturing cells for at least 1 weekpost-thaw before transfecting using the Amaxa P3 Primary Cell 4-DNucleofector Kit (Lonza). Re-feed log-phase growing cells with freshmedium 2 h before transfection. Dissociate to single cells or smallclusters of no more than 10 cells with accutase and gentle resuspension.Count the number of cells needed for nucleofection and spin down at200×g for 5 m. Remove medium completely and resuspend in recommendedvolume of S1-supplemented P3 nucleofection solution. Gently plateelectroporated cells into coated plates in presence of 1× RockInhibitor.

Check transfection success and re-feed daily with regular mTeSR1 mediumbeginning 24 h after nucleofection. Typically, Applicants observegreater than 70% transfection efficiency with Amaxa Nucleofection.Harvest DNA. 48-72 h post transfection, dissociate cells using accutaseand inactivate by adding 5× volume of mTeSR1. Spin cells down at 200×gfor 5 m. Pelleted cells can be directed processed for DNA extractionwith QuickExtract solution. It is recommended to not mechanicallydissociate cells without accutase. It is recommended to not spin cellsdown without inactivating accutase or above the recommended speed; doingso may cause cells to lyse.

Isolation of Clonal Cell Lines by FACS. Timing • 2-3 h Hands-on; 2-3Weeks Expansion

Clonal isolation may be performed 24 h post-transfection by FACS or byserial dilution.

54| Prepare FACS Buffer.

Cells that do not need sorting using colored fluorescence may be sortedin regular D10 medium supplemented with 1× penicillin/streptinomycin. Ifcolored fluorescence sorting is also required, a phenol-free DMEM orDPBS is substituted for normal DMEM. Supplement with 1×penicillin/streptinomycin and filter through a 0.22 um Steriflip filter.

55|Prepare 96 Well Plates.

Applicants added 100 ul of D10 media supplemented with 1×penicillin/streptinomycin per well and prepared the number of plates asneeded for the desired number of clones.

56| Prepare Cells for FACS.

Applicants dissociated cells by aspirating the medium completely andadding 100 ul TrypLE per well of a 24-well plate. Incubate for 5 m andadd 400 ul warm D10 media.

57| Resuspended cells are transferred into a 15 ml Falcon tube andgently triturated 20 times. Recommended to check under the microscope toensure dissociation to single cells.

58| Spin down cells at 200×g for 5 minutes.

59| Applicants aspirated the media, and resuspended the cells in 200 ulof FACS media.

60| Cells are filtered through a 35 um mesh filter into labeled FACStubes. Applicants recommend using the BD Falcon 12×75 mm Tube with CellStrainer cap. Place cells on ice until sorting.

61| Applicants sorted single cells into 96-well plates prepared fromstep 55. Applicants recommend that in one single designated well on eachplate, sort 100 cells as a positive control.

NOTE. The remainder of the cells may be kept and used for genotyping atthe population level to gauge overall modification efficiency.

62| Applicants returned cells into the incubator and allowed them toexpand for 2-3 weeks. 100 ul of warm D10 medium is added 5 d postsorting. Change 100 ul of medium every 3-5 d as necessary.

63| Colonies are inspected for “clonal” appearance 1 week post sorting:rounded colonies radiating from a central point. Mark off wells that areempty or may have been seeded with doublets or multiplets.

64| When cells are more than 60% confluent, Applicants prepared a set ofreplica plates for passaging. 100 ul of D10 medium is added to each wellin the replica plates. Applicants dissociated cells directly bypipetting up and down vigorously 20 times. 20% of the resuspended volumewas plated into the prepared replica plates to keep the clonal lines.Change the medium every 2-3 d thereafter and passage accordingly.

65| Use the remainder 80% of cells for DNA isolation and genotyping.

Optional: Isolation of Clonal Cell Lines by Dilution. Timing • 2-3 hHands-on; 2-3 Weeks Expansion

66| Applicants dissociated cells from 24-well plates as described above.Make sure to dissociate to single cells. A cell strainer can be used toprevent clumping of cells.

67| The number of cells are counted in each condition. Serially diluteeach condition in D10 medium to a final concentration of 0.5 cells per100 ul. For each 96 well plate, Applicants recommend diluting to a finalcount of 60 cells in 12 ml of D10. Accurate count of cell number isrecommended for appropriate clonal dilution. Cells may be recounted atan intermediate serial dilution stage to ensure accuracy.

68| Multichannel pipette was used to pipette 100 ul of diluted cells toeach well of a 96 well plate.

NOTE. The remainder of the cells may be kept and used for genotyping atthe population level to gauge overall modification efficiency.

69| Applicants inspected colonies for “clonal” appearance ˜1 week postplating: rounded colonies radiating from a central point. Mark off wellsthat may have seeded with doublets or multiplets.

70| Applicants returned cells to the incubator and allowed them toexpand for 2-3 weeks. Re-feed cells as needed as detailed in previoussection.

SURVEYOR Assay for CRISPR Cleavage Efficiency. Timing • 5-6 h

Before assaying cleavage efficiency of transfected cells, Applicantsrecommend testing each new SURVEYOR primer on negative (untransfected)control samples through the step of SURVEYOR nuclease digestion usingthe protocol described below. Occasionally, even single-band cleanSURVEYOR PCR products can yield non-specific SURVEYOR nuclease cleavagebands and potentially interfere with accurate indel analysis.

71| Harvest Cells for DNA.

Dissociate cells and spin down at 200×g for 5 m. NOTE. Replica plate atthis stage as needed to keep transfected cell lines.

72| Aspirate the supernatant completely.

73| Applicants used QuickExtract DNA extraction solution according tothe manufacturer's instructions. Applicants typically used 50 ul of thesolution for each well of a 24 well plate and 10 ul for a 96 well plate.

74| Applicants normalized extracted DNA to a final concentration of100-200 ng/ul with ddH2O. Pause point: Extracted DNA may be stored at−20° C. for several months.

75| Set Up the SURVEYOR PCR.

Master mix the following using SURVEYOR primers provided by Applicantsonline/computer algorithm tool:

Final Component: Amount (ul) concentration Herculase II PCR buffer, 5X10 1X dNTP, 100 mM (25 mM each) 1 1 mM SURVEYOR Fwd primer (10 uM) 1 0.2uM SURVEYOR Rev primer (10 uM) 1 0.2 uM Herculase II Fusion polymerase 1MgCl₂ (25 mM) 2 1 mM Distilled water 33 Total 49 (for each reaction)

76| Applicants added 100-200 ng of normalized genomic DNA template fromstep 74 for each reaction.

77| PCR reaction was performed using the following cycling conditions,for no more than 30 amplification cycles:

Cycle number Denature Anneal Extend  1 95° C., 2 min 2-31 95° C., 20 s60° C., 20 s 72° C., 30 s 32 72° C., 3 min

78| Applicants ran 2-5 ul of PCR product on a 1% gel to check forsingle-band product. Although these PCR conditions are designed to workwith most pairs of SURVEYOR primers, some primers may need additionaloptimization by adjusting the template concentration, MgCl₂concentration, and/or the annealing temperature.

79| Applicants purified the PCR reactions using the QIAQuick PCRpurification kit and normalized eluant to 20 ng/ul. Pause point:Purified PCR product may be stored at −20° C.

80| DNA Heteroduplex Formation.

The annealing reaction was set up as follows:

Taq PCR buffer, 10X  2 ul Normalized DNA (20 ng/ul) 18 ul Total volume20 ul

81| Anneal the reaction using the following conditions:

Cycle number Condition 1 95° C., 10 mn 2 95° C.-85° C., −2° C./s 3 85°C., 1 min 4 85° C.-75° C., −0.3° C./s 5 75° C., 1 min 6 75° C.-65° C.,−0.3° C./s 7 65° C., 1 min 8 65° C.-55° C., −0.3° C./s 9 55° C., 1 min10 55° C.-45° C., −0.3° C./s 11 45° C., 1 min 12 45° C.-35° C., −0.3°C./s 13 35° C., 1 min 14 35° C.-25° C., −0.3° C./s 15 25° C., 1 min

82| SURVEYOR Nuclease S Digestion.

Applicants prepared master-mix and added the following components on iceto annealed heteroduplexes from step 81 for a total final volume of 25ul:

Final Component Amount (ul) Concentration MgCl₂ solution, 0.15M 2.5 15mM ddH₂O 0.5 SURVEYOR nuclease S 1 1X SURVEYOR enhancer S 1 1X Total 5

83| Vortex well and spin down. Incubate the reaction at 42° C. for 1 h.

84| Optional: 2 ul of the Stop Solution from the SURVEYOR kit may beadded. Pause point. The digested product may be stored at −20° C. foranalysis at a later time.

85| Visualize the SURVEYOR Reaction.

SURVEYOR nuclease digestion products may be visualized on a 2% agarosegel. For better resolution, products may be run on a 4-20% gradientPolyacrylamide TBE gel. Applicants loaded 10 ul of product with therecommended loading buffer and ran the gel according to manufacturer'sinstructions. Typically, Applicants run until the bromophenol blue dyehas migrated to the bottom of the gel. Include DNA ladder and negativecontrols on the same gel.

86| Applicants stained the gel with 1×SYBR Gold dye diluted in TBE. Thegel was gently rocked for 15 m.

87| Applicants imaged the gel using a quantitative imaging systemwithout overexposing the bands. The negative controls should have onlyone band corresponding to the size of the PCR product, but may haveoccasionally non-specific cleavage bands of other sizes. These will notinterfere with analysis if they are different in size from targetcleavage bands. The sum of target cleavage band sizes, provided byApplicants online/computer algorithm tool, should be equal to the sizeof the PCR product.

88| Estimate the Cleavage Intensity.

Applicants quantified the integrated intensity of each band using ImageJor other gel quantification software.

89| For each lane, Applicants calculated the fraction of the PCR productcleaved (f_(cut)) using the following formula: f_(cut)=(b+c)/(a+b+c),where a is the integrated intensity of the undigested PCR product and band c are the integrated intensities of each cleavage product. 90|Cleavage efficiency may be estimated using the following formula, basedon the binomial probability distribution of duplex formation:

indel (%)=100×(1−√{square root over ((1f _(cut)))})  91|

Sanger Sequencing for Assessing CRISPR Cleavage Efficiency. Timing • 3 d

Initial steps are identical to Steps 71-79 of the SURVEYOR assay. Note:SURVEYOR primers may be used for Sanger sequencing if appropriaterestriction sites are appended to the Forward and Reverse primers. Forcloning into the recommended pUC19 backbone, EcoRI may be used for theFwd primer and HindIII for the Rev primer.

92| Amplicon Digestion.

Set up the digestion reaction as follows:

Component Amount (ul) Fast Digest buffer, 10X 3 FastDigest EcoRI 1FastDigest HindIII 1 Normalized DNA (20 ng/ul) 10 ddH₂O 15 Total volume30

93| pUC19 backbone digestion. Set up the digestion reaction as follows:

Component Amount (ul) Fast Digest buffer, 10X 3 FastDigest EcoRI 1FastDigest HindIII 1 FastAP Alkaline Phosphatase 1 pUC19 vector (200ng/ul) 5 ddH₂O 20 Total volume 30

94| Applicants purified the digestion reactions using the QIAQuick PCRpurification kit. Pause point: Purified PCR product may be stored at−20° C.

95| Applicants ligated the digested pUC19 backbone and Sanger ampliconsat a 1:3 vector:insert ratio as follows:

Component Amount (ul) Digested pUC19 x (50 ng) Digested insert x (1:3vector:insert molar ratio) T7 ligase  1 2X Rapid Ligation Buffer 10ddH₂O x Total volume 20

96| Transformation.

Applicants transformed the PlasmidSafe-treated plasmid into a competentE. coli strain, according to the protocol supplied with the cells.Applicants recommend Stb13 for quick transformation. Briefly, 5 ul ofthe product from step 95 is added into 20 ul of ice-cold chemicallycompetent Stb13 cells, incubated on ice for 10 m, heat shocked at 42° C.for 30 s, returned immediately to ice for 2 m, 100 ul of SOC medium isadded, and plated onto an LB plate containing 100 ug/ml ampicillin. Thisis incubated overnight at 37° C.

97| Day 2: Applicants inspected plates for colony growth. Typically,there are no colonies on the negative control plates (ligation ofEcoRI-HindIII digested pUC19 only, no Sanger amplicon insert), and tensto hundreds of colonies on the pUC19-Sanger amplicon cloning plates.

98| Day 3: Applicants isolated plasmid DNA from overnight cultures usinga QIAprep Spin miniprep kit according to the manufacturer'sinstructions.

99| Sanger Sequencing.

Applicants verified the sequence of each colony by sequencing from thepUC19 backbone using the pUC19-For primer. Applicants referenced thesequencing results against the expected genomic DNA sequence to checkfor the presence of Cas9-induced NHEJ mutations. % editing efficiency=(#modified clones)/(# total clones). It is important to pick a reasonablenumber of clones (>24) to generate accurate modification efficiencies.

Genotyping for Microdeletion. Timing • 2-3 d Hands on; 2-3 WeeksExpansion

100| Cells were transfected as described above with a pair of sgRNAstargeting the region to be deleted.

101| 24 h post-transfection, clonal lines are isolated by FACS or serialdilution as described above.

102| Cells are expanded for 2-3 weeks.

103| Applicants harvested DNA from clonal lines as described above using10 ul QuickExtract solution and normalized genomic DNA with ddH₂O to afinal concentration of 50-100 ng/ul.

104| PCR Amplify the Modified Region.

The PCR reaction is set up as follows:

Final Component: Amount (ul) concentration Herculase II PCR buffer, 5X10 1X dNTP, 100 mM (25 mM each) 1 1 mM Out Fwd primer (10 uM) 1 0.2 uMOut Rev primer (10 uM) 1 0.2 uM Herculase II Fusion polymerase 1 MgCl2(25 mM) 2 1 mM ddH₂O 32 Total 48 (for each reaction)

Note: if deletion size is more than 1 kb, set up a parallel set of PCRreactions with In-Fwd and In-Rev primers to screen for the presence ofthe wt allele.

105| To screen for inversions, a PCR reaction is set up as follows:

Final Component: Amount (ul) concentration Herculase II PCR buffer, 5X10 1X dNTP, 100 mM (25 mM each) 1 1 mM Out Fwd or Out-Rev primer (10 uM)1 0.2 uM In Fwd or In-Rev primer (10 uM) 1 0.2 uM Herculase II Fusionpolymerase 1 MgCl₂ (25 mM) 2 1 mM ddH₂O 32 Total 48 (for each reaction)

Note: primers are paired either as Out-Fwd+In Fwd, or Out-Rev+In-Rev.

106| Applicants added 100-200 ng of normalized genomic DNA template fromstep 103 for each reaction.

107| PCR reaction was performed using the following cycling conditions:

Cycle number Denature Anneal Extend  1 95° C., 2 min 2-31 95° C., 20 s60° C., 20 s 72° C., 30 s 32 72° C., 3 m

108| Applicants run 2-5 ul of PCR product on a 1-2% gel to check forproduct. Although these PCR conditions are designed to work with mostprimers, some primers may need additional optimization by adjusting thetemplate concentration, MgCl₂ concentration, and/or the annealingtemperature.

Genotyping for Targeted Modifications Via HDR. Timing • 2-3 d, 2-3 hHands on

109| Applicants harvested DNA as described above using QuickExtractsolution and normalized genomic DNA with TE to a final concentration of100-200 ng/ul.

110| PCR Amplify the Modified Region.

The PCR reaction is set up as follows:

Final Component: Amount (ul) concentration Herculase II PCR buffer, 5X10 1X dNTP, 100 mM (25 mM each) 1 1 mM HDR Fwd primer (10 uM) 1 0.2 uMHDR Rev primer (10 uM) 1 0.2 uM Herculase II Fusion polymerase 1 MgCl₂(25 mM) 2 1 mM ddH₂O 33 Total 49 (for each reaction)

111| Applicants added 100-200 ng of genomic DNA template from step 109for each reaction and run the following program.

Cycle number Denature Anneal Extend  1 95° C., 2 min 2-31 95° C., 20 s60° C., 20 s 72° C., 30-60 s per kb 32 72° C., 3 min

112| Applicants ran 5 ul of PCR product on a 0.8-1% gel to check forsingle-band product. Primers may need additional optimization byadjusting the template concentration, MgCl₂ concentration, and/or theannealing temperature.

113| Applicants purified the PCR reactions using the QIAQuick PCRpurification kit.

114| In the HDR example, a HindIII restriction site is inserted into theEMX1 gene. These are detected by a restriction digest of the PCRamplicon:

Component Amount (ul) Purified PCR amplicon (200-300 ng) x F.D. buffer,Green 1 HindIII 0.5 ddH2O x Total 10

i. The DNA is digested for 10 m at 37° C.:

ii. Applicants ran 10 ul of the digested product with loading dye on a4-20% gradient polyacrylamide TBE gel until the xylene cyanol band hadmigrated to the bottom of the gel.

iii. Applicants stained the gel with 1×SYBR Gold dye while rocking for15 m.

iv. The cleavage products are imaged and quantified as described abovein the SURVEYOR assay section. HDR efficiency is estimated by theformula: (b+c)/(a+b+c), where a is the integrated intensity for theundigested HDR PCR product, and b and c are the integrated intensitiesfor the HindIII-cut fragments.

115| Alternatively, purified PCR amplicons from step 113 may be clonedand genotyped using Sanger sequencing or NGS.

Deep Sequencing and Off-Target Analysis • Timing 1-2 d

The online CRISPR target design tool generates candidate genomicoff-target sites for each identified target site. Off-target analysis atthese sites can be performed by SURVEYOR nuclease assay, Sangersequencing, or next-generation deep sequencing. Given the likelihood oflow or undetectable modification rates at many of these sites,Applicants recommend deep sequencing with the Illumina Miseq platformfor high sensitivity and accuracy. Protocols will vary with sequencingplatform; here, Applicants briefly describe a fusion PCR method forattaching sequencing adapters.

116| Design Deep Sequencing Primers.

Next-generation sequencing (NGS) primers are designed for shorteramplicons, typically in the 100-200 bp size range. Primers may bemanually designed using NCBI Primer-Blast or generated with onlineCRISPR target design tools (website at genome-engineering. org/tools).

117| Harvest genomic DNA from Cas9-targeted cells. NormalizeQuickExtract genomic DNA to 100-200 ng/ul with ddH2O.

118| Initial Library Preparation PCR.

Using the NGS primers from step 116, prepare the initial librarypreparation PCR

Final Component: Amount (ul) concentration Herculase II PCR buffer, 5X10 1X dNTP, 100 mM (25 mM each) 1 1 mM NGS Fwd primer (10 uM) 1 0.2 uMNGS Rev primer (10 uM) 1 0.2 uM Herculase II Fusion polymerase 1 MgCl2(25 mM) 2 1 mM ddH2O 33 Total 49 (for each reaction)

119| Add 100-200 ng of normalized genomic DNA template for eachreaction.

120| Perform PCR reaction using the following cycling conditions, for nomore than 20 amplification cycles:

Cycle number Denature Anneal Extend  1 95° C., 2 min 2-21 95° C., 20 s60° C., 20 s 72° C., 15 s 22 72° C., 3 min

121| Run 2-5 ul of PCR product on a 1% gel to check for single-bandproduct. As with all genomic DNA PCRs, NGS primers may requireadditional optimization by adjusting the template concentration, MgCl₂concentration, and/or the annealing temperature.

122| Purify the PCR reactions using the QIAQuick PCR purification kitand normalize eluant to 20 ng/ul. Pause point: Purified PCR product maybe stored at −20° C.

123|Nextera XT DNA Sample Preparation Kit.

Following the manufacturer's protocol, generate Miseq sequencing-readylibraries with unique barcodes for each sample.

124| Analyze Sequencing Data.

Off-target analysis may be performed through read alignment programssuch as ClustalW, Geneious, or simple sequence analysis scripts.

Timing

Steps 1-2 Design and synthesis of sgRNA oligos and ssODNs: 1-5 d,variable depending on supplier

Steps 3-5 Construction of CRISPR plasmid or PCR expression cassette: 2 hto 3 d

Steps 6-53 Transfection into cell lines: 3 d (1 h hands-on time)

Steps 54-70 Optional derivation of clonal lines: 1-3 weeks, variabledepending on cell type

Steps 71-91 Functional validation of NHEJ via SURVEYOR: 5-6 h

Steps 92-124 Genotyping via Sanger or next-gen deep sequencing: 2-3 d(3-4 h hands on time)

Addressing Situations Concerning Herein Examples

Situation Solution No amplification of Titrate U6-template concentrationsgRNA SURVEYOR or HDR PCR Titrate MgCl2; normalize and titrate templatedirty or no amplification concentration; annealing temp gradient;redesign primers Unequal amplification of Set up separate PCRs to detectwildtype and deletion alleles in microdeletion alleles; Redesign primerswith similar sized amplicons PCRs Colonies on negative Increase BbsI;increase Golden Gate reaction cycle control plate number, cut PX330separately with Antarctic Phosphate treatment No sgRNA sequences orScreen additional colonies wrong sequences Low lipofectamine Check cellhealth and density; titrate DNA; add GFP transfection efficiencytransfection control Low nucleofection Check cell health and density;titrate DNA; suspend to transfection efficiency single cell Clumps or nocells after Filter cells before FACS; dissociate to single cells; FACSresuspend in appropriate density Clumps or no cells in serial Recountcells; dissociate to single cells and filter through dilution strainer;check serial dilution High SURVEYOR Redesign primers to prime fromdifferent locations background on negative sample Dirty SURVEYOR resultPurify PCR product; reduce input DNA; reduce 42° C. on gel incubation to30 m No SURVEYOR cleavage Purify and normalize PCR product; re-annealwith TaqB buffer; Redesign sgRNAs; sequence verify Cas9 on px330backbone Samples do not sink in Supplement with MgCl2 to a finalconcentration of 15 mM TBE acrylamide gel or add loading buffercontaining glycerol

Discussion

CRISPR-Cas may be easily multiplexed to facilitate simultaneousmodification of several genes and mediate chromosomal microdeletions athigh efficiencies. Applicants used two sgRNAs to demonstratesimultaneous targeting of the human GRIN2B and DYRK1A loci atefficiencies of up to 68% in HEK293FT cells. Likewise, a pair of sgRNAsmay be used to mediate microdeletions, such as excision of an exon,which can be genotyped by PCR on a clonal level. Note that the preciselocation of exon junctions can vary. Applicants also demonstrated theuse of ssODNs and targeting vector to mediate HDR with both wildtype andnickase mutant of Cas9 in HEK 293FT and HUES9 cells (FIG. 12). Note thatApplicants have not been able to detect HDR in HUES9 cells using theCas9 nickase, which may be due to low efficiency or a potentialdifference in repair activities in HUES9 cells. Although these valuesare typical, there is some variability in the cleavage efficiency of agiven sgRNA, and on rare occasions certain sgRNAs may not work forreasons yet unknown. Applicants recommend designing two sgRNAs for eachlocus, and testing their efficiencies in the intended cell type.

Example 26 NLSs

Cas9 Transcriptional Modulator: Applicants set out to turn the Cas9/gRNACRISPR system into a generalized DNA binding system in which functionsbeyond DNA cleavage can be executed. For instance, by fusing functionaldomain(s) onto a catalytically inactive Cas9 Applicants have impartednovel functions, such as transcriptional activation/repression,methylation/demethylation, or chromatin modifications. To accomplishthis goal Applicants made a catalytically inactive Cas9 mutant bychanging two residues essential for nuclease activity, D10 and H840, toalanine. By mutating these two residues the nuclease activity of Cas9 isabolished while maintaining the ability to bind target DNA. Thefunctional domains Applicants decided to focus on to test Applicants'hypothesis are the transcriptional activator VP64 and thetranscriptional repressors SID and KRAB.

Cas9 Nuclear localization: Applicants hypothesized that the mosteffective Cas9 transcriptional modulator would be strongly localized tothe nucleus where it would have its greatest influence on transcription.Moreover, any residual Cas9 in the cytoplasm could have unwantedeffects. Applicants determined that wild-type Cas9 does not localizeinto the nucleus without including multiple nuclear localization signals(NLSs) (although a CRISPR system need not have one or more NLSs butadvantageously has at least one or more NLS(s)). Because multiple NLSsequences were required it was reasoned that it is difficult to get Cas9into the nucleus and any additional domain that is fused to Cas9 coulddisrupt the nuclear localization. Therefore, Applicants made fourCas9-VP64-GFP fusion constructs with different NLS sequences(pXRP02-pLenti2-EF1a-NLS-hSpCsn1(10A,840A)-NLS-VP64-EGFP,pXRPO4-pLenti2-EF1a-NLS-hSpCsn1(10A,840A)-NLS-VP64-2A-EGFP-NLS,pXRP06-pLenti2-EF1a-NLS-EGFP-VP64-NLS-hSpCsn1(10A,840A)-NLS,pXRP08-pLenti2-EF1a-NLS-VP64-NLS-hSpCsn1(10A,840A)-NLS-VP64-EGFP-NLS).These constructs were cloned into a lenti backbone under the expressionof the human EF1a promoter. The WPRE element was also added for morerobust protein expression. Each construct was transfected into HEK 293FTcells using Lipofectame 2000 and imaged 24 hours post-transfection. Thebest nuclear localization is obtained when the fusion proteins have NLSsequences on both the N- and C-term of the fusion protein. The highestobserved nuclear localization occurred in the construct with four NLSelements.

To more robustly understand the influence of NLS elements on Cas9Applicants made 16 Cas9-GFP fusions by adding the same alpha importinNLS sequence on either the N- or C-term looking at zero to three tandemrepeats. Each construct was transfected into HEK 293FT cells usingLipofectame 2000 and imaged 24 hours post-transfection. Notably, thenumber of NLS elements does not directly correlate with the extent ofnuclear localization. Adding an NLS on the C-term has a greaterinfluence on nuclear localization than adding on the N-term.

Cas9 Transcriptional Activator: Applicants functionally tested theCas9-VP64 protein by targeting the Sox2 locus and quantifyingtranscriptional activation by RT-qPCR. Eight DNA target sites werechosen to span the promoter of Sox2. Each construct was transfected intoHEK 293FT cells using Lipofectame 2000 and 72 hours post-transfectiontotal RNA was extracted from the cells. 1 ug of RNA was reversetranscribed into cDNA (qScript Supermix) in a 40 ul reaction. 2 ul ofreaction product was added into a single 20 ul TaqMan assay qPCRreaction. Each experiment was performed in biological and technicaltriplicates. No RT control and no template control reactions showed noamplification. Constructs that do not show strong nuclear localization,pXRPO2 and pXRPO4, result in no activation. For the construct that didshow strong nuclear localization, pXRP08, moderate activation wasobserved. Statistically significant activation was observed in the caseof guide RNAs Sox2.4 and Sox2.5.

Example 27 In Vivo Mouse Data

Material and Reagents

Herculase II fusion polymerase (Agilent Technologies, cat. no. 600679)10× NEBuffer 4 (NEB, cat. No. B7004S)BsaI HF (NEB, cat. No. R3535S)T7 DNA ligase (Enzymatics, cat. no. L602L)Fast Digest buffer, 10× (ThermoScientific, cat. No. B64)FastDigest NotI (ThermoScientific, cat. No. FD0594)FastAP Alkaline Phosphatase (ThermoScientific, cat. No. EF0651)Lipofectamine2000 (Life Technologies, cat. No. 11668-019)Trypsin (Life Technologies, cat. No. 15400054)Forceps #4 (Sigma, cat. No. Z168777-1EA)Forceps #5 (Sigma, cat. No. F6521-1EA)10× Hank's Balanced Salt Solution (Sigma, cat. No. H4641-500ML)Penicillin/Streptomycin solution (Life Technologies, cat. No. P4333)Neurobasal (Life Technologies, cat. No. 21103049)B27 Supplement (Life Technologies, cat. No. 17504044)L-glutamine (Life Technologies, cat. No. 25030081)Glutamate (Sigma, cat. No. RES5063G-A7)β-mercaptoethanol (Sigma, cat. No. M6250-100ML)HA rabbit antibody (Cell Signaling, cat. No. 3724S)LIVE/DEAD® Cell Imaging Kit (Life Technologies, cat. No. R37601)30G World Precision Instrument syringe (World Precision Instruments,cat. No. NANOFIL)Stereotaxic apparatus (Kopf Instruments)UltraMicroPump3 (World Precision Instruments, cat. No. UMP3-4)Sucrose (Sigma, cat. No. S7903)Calcium chloride (Sigma, cat. No. C1016)Magnesium acetate (Sigma, cat. No. M0631)

Tris-HCl (Sigma, cat. no T5941)

EDTA (Sigma, cat. No. E6758)NP-40 (Sigma, cat. No. NP40)Phenylmethanesulfonyl fluoride (Sigma, cat. No. 78830)Magnesium chloride (Sigma, cat. No. M8266)Potassium chloride (Sigma, cat. No. P9333)β-glycerophosphate (Sigma, cat. No. G9422)Glycerol (Sigma, cat. No. G9012)Vybrant® DyeCycle™ Ruby Stain (Life technologies, cat. No. S4942)FACS Aria Flu-act-cell sorter (Koch Institute of MIT, Cambridge US)DNAeasy Blood & Tissue Kit (Qiagen, cat. No. 69504)

Procedure

Constructing gRNA Multiplexes for Using In Vivo in the Brain Applicantsdesigned and PCR amplified single gRNAs targeting mouse TET and DNMTfamily members (as described herein) Targeting efficiency was assessedin N2a cell line (FIG. 15). To obtain simultaneous modification ofseveral genes in vivo, efficient gRNA was multiplexed in AAV-packagingvector (FIG. 16). To facilitate further analysis of system efficiencyapplicants added to the system expression cassette consistent ofGFP-KASH domain fusion protein under control of human Synapsin Ipromoter (FIG. 16). This modification allows for further analysis ofsystem efficiency in neuronal population (more detail procedure insection Sorting nuclei and in vivo results).

All 4 parts of the system were PCR amplified using Herculase II Fusionpolymerase using following primers:

1st U6 Fw: (SEQ ID NO: 185)gagggtctcgtccttgcggccgcgctagcgagggcctatttcccatga ttc 1st gRNA Rv: (SEQID NO: 186) ctcggtctcggtAAAAAAgcaccgactcggtgccactttttcaagttgataacggactagccttattttaacttgctaTTTCtagctctaaaacNNNNNNNNNNNNNNNNNNNNGGTGTTTCGTCCTTTCCAC 2nd U6 Fw: (SEQ ID NO: 187)gagggtctcTTTaccggtgagggcctatttcccatgattcc 2nd gRNA Rv: (SEQ ID NO: 188)ctcggtctcctcAAAAAAgcaccgactcggtgccactttttcaagttgataacggactagccttattttaacttgctaTTTCtagctctaaaacNNNNNNNNNNNNNNNNNNNNGGTGTTTCGTCCTTTCCAC 3rd U6 Fw: (SEQ ID NO: 189)gagggtctcTTTgagctcgagggcctatttcccatgattc 3rd gRNA Rv: (SEQ ID NO: 190)ctcggtctcgcgtAAAAAAgcaccgactcggtgccactttttcaagttg ataacggactagccttattttaacttgctaTTTCtagctctaaaacNNNNNNNNNNNNNNNNNNNNGGTGTTTCGTCCTTTCCA hSyn_GFP-kash Fw: (SEQ ID NO: 191)gagggtctcTTacgcgtgtgtctagac hSyn_GFP-kash Rv: (SEQ ID NO: 192)ctcggtctcAaggaCAGGGAAGGGAGCAGTGGTTCACGCCTGTAATCC CAGCAATTTGGGAGGCCAAGGTGGGTAGATCACCTGAGATTAGG AGTTGC (NNNNNNNNNNNNNNNNNNNN is areverse compliment targeted genomic sequence)

Applicants used Golden Gate strategy to assemble all parts (1:1molecular ratio) of the system in a single step reaction:

1^(st) U6_gRNA 18 ng 2^(nd) U6_gRNA 18 ng 3^(rd) U6_gRNA 18 ngSyn_GFP-kash 100 ng 10x NEBuffer 4 1.0 μl 10x BSA 1.0 μl 10 mM ATP 1.0μl BsaI HF 0.75 μl T7 ligase 0.25 μl ddH₂O 10 μl Cycle number Condition1-50 37° C. for 5 m, 21° C. for 5 m

Golden Gate reaction product was PCR amplified using Herculase II fusionpolymerase and following primers:

Fw (SEQ ID NO: 193) 5′ cctgtccttgcggccgcgctagcgagggcc Rv (SEQ ID NO:194) 5′ cacgcggccgcaaggacagggaagggagcag

PCR product was cloned into AAV backbone, between ITR sequences usingNotI restriction sites:

PCR product digestion:

Fast Digest buffer, 10X 3 μl FastDigest NotI 1 μl DNA 1 μg ddH₂O up to30 μl

AAV backbone digestion:

Fast Digest buffer, 10X 3 μl FastDigest NotI 1 μl FastAP AlkalinePhosphatase 1 μl AAV backbone 1 μg ddH₂O up to 30 μl

After 20 min incubation in 37° C. samples were purified using QIAQuickPCR purification kit. Standardized samples were ligated at a 1:3vector:insert ratio as follows:

Digested pUC19 50 ng Digested insert 1:3 vector:insert molar ratio T7ligase 1 μl 2X Rapid Ligation Buffer 5 μl ddH₂O up to 10 μl

After transformation of bacteria with ligation reaction product,applicants confirmed obtained clones with Sanger sequencing.

Positive DNA clones were tested in N2a cells after co-transfection withCas9 construct (FIGS. 17 and 18).

Design of New Cas9 Constructs for AAV Delivery

AAV delivery system despite its unique features has packinglimitation—to successfully deliver expressing cassette in vivo it has tobe in size <then 4.7 kb. To decrease the size of SpCas9 expressingcassette and facilitate delivery applicants tested several alteration:different promoters, shorter polyA signal and finally a smaller versionof Cas9 from Staphylococcus aureus (SaCas9) (FIGS. 19 and 20). Alltested promoters were previously tested and published to be active inneurons, including mouse Mecp2 (Gray et al., 2011), rat Maplb andtruncated rat Map1b (Liu and Fischer, 1996). Alternative synthetic polyAsequence was previously shown to be functional as well (Levitt et al.,1989; Gray et al., 2011). All cloned constructs were expressed in N2acells after transfection with Lipofectamine 2000, and tested withWestern blotting method (FIG. 21).

Testing AAV Multiplex System in Primary Neurons

To confirm functionality of developed system in neurons, Applicants useprimary neuronal cultures in vitro. Mouse cortical neurons was preparedaccording to the protocol published previously by Banker and Goslin(Banker and Goslin, 1988).

Neuronal cells are obtained from embryonic day 16. Embryos are extractedfrom the euthanized pregnant female and decapitated, and the heads areplaced in ice-cold HBSS. The brains are then extracted from the skullswith forceps (#4 and #5) and transferred to another change of ice-coldHBSS. Further steps are performed with the aid of a stereoscopicmicroscope in a Petri dish filled with ice-cold HBSS and #5 forceps. Thehemispheres are separated from each other and the brainstem and clearedof meninges. The hippocampi are then very carefully dissected and placedin a 15 ml conical tube filled with ice-cold HBSS. Cortices that remainafter hippocampal dissection can be used for further cell isolationusing an analogous protocol after removing the brain steam residuals andolfactory bulbs. Isolated hippocampi are washed three times with 10 mlice-cold HBSS and dissociated by 15 min incubation with trypsin in HBSS(4 ml HBSS with the addition of 10 μl 2.5% trypsin per hippocampus) at37° C. After trypsinization, the hippocampi are very carefully washedthree times to remove any traces of trypsin with HBSS preheated to 37°C. and dissociated in warm HBSS. Applicants usually dissociate cellsobtained from 10-12 embryos in 1 ml HBSS using 1 ml pipette tips anddilute dissociated cells up to 4 ml. Cells are plated at a density of250 cells/mm2 and cultured at 37° C. and 5% CO2 for up to 3 week

HBSS

435 ml H₂O 50 ml 10× Hank's Balanced Salt Solution 16.5 ml 0.3M HEPES pH7.3

5 ml penicillin-streptomycin solutionFilter (0.2 μm) and store 4° C.

Neuron Plating Medium (100 ml)

97 ml Neurobasal 2 ml B27 Supplement

1 ml penicillin-streptomycin solution250 μl glutamine125 μl glutamate

Neurons are transduced with concentrated AAV1/2 virus or AAV1 virus fromfiltered medium of HEK293FT cells, between 4-7 days in culture and keepfor at least one week in culture after transduction to allow fordelivered gene expression.

AAV-Driven Expression of the System

Applicants confirmed expression of SpCas9 and SaCas9 in neuronalcultures after AAV delivery using Western blot method (FIG. 24). Oneweek after transduction neurons were collected in NuPage SDS loadingbuffer with β-mercaptoethanol to denaturate proteins in 95° C. for 5min. Samples were separated on SDS PAGE gel and transferred on PVDFmembrane for WB protein detection. Cas9 proteins were detected with HAantibody.

Expression of Syn-GFP-kash from gRNA multiplex AAV was confirmed withfluorescent microscopy (FIG. 32).

Toxicity

To assess the toxicity of AAV with CRISPR system Applicants testedoverall morphology of neurons one week after virus transduction (FIG.27). Additionally, Applicants tested potential toxicity of designedsystem with the LIVE/DEAD® Cell Imaging Kit, which allows to distinguishlive and dead cells in culture. It is based on the presence ofintracellular esterase activity (as determined by the enzymaticconversion of the non-fluorescent calcein AM to the intensely greenfluorescent calcein). On the other hand, the red, cell-impermeantcomponent of the Kit enters cells with damaged membranes only and bindto DNA generating fluorescence in dead cells. Both flourophores can beeasily visualized in living cells with fluorescent microscopy.AAV-driven expression of Cas9 proteins and multiplex gRNA constructs inthe primary cortical neurons was well tolerated and not toxic (FIGS. 25and 26), what indicates that designed AAV system is suitable for in vivotests.

Virus Production

Concentrated virus was produced according to the methods described inMcClure et al., 2011. Supernatant virus production occurred in HEK293FTcells.

Brain Surgeries

For viral vector injections 10-15 week old male C57BL/6N mice wereanesthetized with a Ketamine/Xylazine cocktail (Ketamine dose of 100mg/kg and Xylazine dose of 10 mg/kg) by intraperitoneal injection.Intraperitonial administration of Buprenex was used as a pre-emptiveanalgesic (1 mg/kg). Animals were immobilized in a Kopf stereotaxicapparatus using intra-aural positioning studs and tooth bar to maintainan immobile skull. Using a hand-held drill, a hole (1-2 mm) at −3.0 mmposterior to Bregma and 3.5 mm lateral for injection in the CA1 regionof the hippocampus was made. Using 30G World Precision Instrumentsyringe at a depth of 2.5 mm, the solution of AAV viral particles in atotal volume of 1 ul was injected. The injection was monitored by a‘World Precision Instruments UltraMicroPump3’ injection pump at a flowrate of 0.5 ul/min to prevent tissue damage. When the injection wascomplete, the injection needle was removed slowly, at a rate of 0.5mm/min. After injection, the skin was sealed with 6-0 Ethilon sutures.Animals were postoperatively hydrated with 1 mL lactated Ringer's(subcutaneous) and housed in a temperature controlled (37° C.)environment until achieving an ambulatory recovery. 3 weeks aftersurgery animals were euthanized by deep anesthesia followed by tissueremoval for nuclei sorting or with 4% paraformaldehyde perfusion forimmunochemistry.

Sorting Nuclei and In Vivo Results

Applicants designed a method to specifically genetically tag the gRNAtargeted neuronal cell nuclei with GFP for Fluorescent Activated CellSorting (FACS) of the labeled cell nuclei and downstream processing ofDNA, RNA and nuclear proteins. To that purpose the applicants' multiplextargeting vector was designed to express both a fusion protein betweenGFP and the mouse nuclear membrane protein domain KASH (Starr D A, 2011,Current biology) and the 3 gRNAs to target specific gene loci ofinterest (FIG. 16). GFP-KASH was expressed under the control of thehuman Synapsin promoter to specifically label neurons. The amino acid ofthe fusion protein GFP-KASH was:

(SEQ ID NO: 195) MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKSGLRSREEEEETDSRMPHLDSPGSSQPRRSFLSRVIRAALPLQLLLLLLLLLACLLPASEDDYSCTQANNFARSFYPMLRYTNGPPPT

One week after AAV1/2 mediated delivery into the brain a robustexpression of GFP-KASH was observed. For FACS and downstream processingof labeled nuclei, the hippocampi were dissected 3 weeks after surgeryand processed for cell nuclei purification using a gradientcentrifugation step. For that purpose the tissue was homogenized in 320mM Sucrose, 5 mM CaCl, 3 mM Mg(Ac)2, 10 mM Tris pH 7.8, 0.1 mM EDTA,0.1% NP40, 0.1 mM Phenylmethanesulfonyl fluoride (PMSF), 1 mMβ-mercaptoethanol using 2 ml Dounce homogenizer (Sigma) The homogenisatewas centrifuged on a 25% to 29% Optiprep® gradient according to themanufacture's protocol for 30 min at 3.500 rpm at 4° C. The nuclearpellet was resuspended in 340 mM Sucrose, 2 mM MgCl2, 25 mM KCl, 65 mMglycerophosphate, 5% glycerol, 0.1 mM PMSF, 1 mM β-mercaptoethanol andVybrant® DyeCycle™ Ruby Stain (Life technologies) was added to labelcell nuclei (offers near-infrared emission for DNA). The labeled andpurified nuclei were sorted by FACS using an Aria Flu-act-cell sorterand BDFACS Diva software. The sorted GFP+ and GFP− nuclei were finallyused to purify genomic DNA using DNAeasy Blood & Tissue Kit (Qiagen) forSurveyor assay analysis of the targeted genomic regions. The sameapproach can be easily used to purify nuclear RNA or protein fromtargeted cells for downstream processing. Due to the 2-vector system(FIG. 16) the applicants using in this approach efficient Cas9 mediatedDNA cleavage was expected to occur only in a small subset of cells inthe brain (cells which were co-infected with both the multiplextargeting vector and the Cas9 encoding vector). The method describedhere enables the applicants to specifically purify DNA, RNA and nuclearproteins from the cell population expressing the 3 gRNAs of interest andtherefore are supposed to undergo Cas9 mediated DNA cleavage. By usingthis method the applicants were able to visualize efficient DNA cleavagein vivo occurring only in a small subset of cells.

Essentially, what Applicants have shown here is targeted in vivocleavage. Furthermore, Applicants used a multiple approach, with severaldifferent sequences targeted at the same time, but independently.Presented system can be applied for studying brain pathologic conditions(gene knock out, e.g. Parkinson disease) and also open a field forfurther development of genome editing tools in the brain. By replacingnuclease activity with gene transcription regulators or epigeneticregulators it will be possible to answer whole spectrum of scientificquestion about role of gene regulation and epigenetic changes in thebrain in not only in the pathologic conditions but also in physiologicalprocess as learning and memory formation. Finally, presented technologycan be applied in more complex mammalian system as primates, what allowsto overcome current technology limitations.

Example 28 Model Data

Several disease models have been specifically investigated. Theseinclude de novo autism risk genes CHD8, KATNAL2, and SCN2A; and thesyndromic autism (Angelman Syndrome) gene UBE3A. These genes andresulting autism models are of course preferred, but show that theinvention may be applied to any gene and therefore any model ispossible.

Applicants have made these cells lines using Cas9 nuclease in humanembryonic stem cells (hESCs). The lines were created by transienttransfection of hESCs with Cbh-Cas9-2A-EGFP and pU6-sgRNA. Two sgRNAsare designed for each gene targeting most often the same exons in whichpatient nonsense (knock-out) mutations have been recently described fromwhole exome sequencing studies of autistic patients. The Cas9-2A-EGFPand pU6 plasmids were created specifically for this project.

Example 29 AAV Production System or Protocol

An AAV production system or protocol that was developed for, and worksparticularly well with, high through put screening uses is providedherein, but it has broader applicability in the present invention aswell. Manipulating endogenous gene expression presents variouschallenges, as the rate of expression depends on many factors, includingregulatory elements, mRNA processing, and transcript stability. Toovercome this challenge, Applicants developed an adeno-associated virus(AAV)-based vector for the delivery. AAV has an ssDNA-based genome andis therefore less susceptible to recombination.

AAV1/2 (serotype AAV1/2, i.e., hybrid or mosaic AAV1/AAV2 capsid AAV)heparin purified concentrated virus protocol

Media: D10+HEPES

500 ml bottle DMEM high glucose+Glutamax (GIBCO)50 ml Hyclone FBS (heat-inactivated) (Thermo Fischer)5.5 ml HEPES solution (1M, GIBCO)Cells: low passage HEK293FT (passage <10 at time of virus production,thaw new cells of passage 2-4 for virus production, grow up for 3-5passages)

Transfection Reagent: Polyethylenimine (PEI) “Max”

Dissolve 50 mg PEI “Max” in 50 ml sterile Ultrapure H₂O

Adjust pH to 7.1

Filter with 0.22 um fliptop filterSeal tube and wrap with parafilmFreeze aliquots at −20° C. (for storage, can also be used immediately)

Cell Culture

Culture low passage HEK293FT in D10+HEPESPassage everyday between 1:2 and 1:2.5Advantageously do not allow cells to reach more than 85% confluency

For T75

Warm 10 ml HBSS (—Mg2+, —Ca2+, GIBCO)+1 ml TrypLE Express (GIBCO) perflask to 37° C. (Waterbath)

Aspirate media fully

Add 10 ml warm HBSS gently (to wash out media completely)

Add 1 ml TrypLE per Flask

Place flask in incubator (37° C.) for 1 min

Rock flask to detach cells

Add 9 ml D10+HEPES media (37° C.)

Pipette up and down 5 times to generate single cell suspension

Split at 1:2-1:2.5 (12 ml media for T75) ratio (if cells are growingmore slowly, discard and thaw a new batch, they are not in optimalgrowth)

transfer to T225 as soon as enough cells are present (for ease ofhandling large amounts of cells)

AAV Production (5*15 cm Dish Scale Per Construct):

Plate 10 million cells in 21.5 ml media into a 15 cm dishIncubate for 18-22 hours at 37° C.Transfection is ideal at 80% confluence

Per Plate

Prewarm 22 ml media (D10+HEPES)

Prepare Tube with DNA Mixture (Use Endofree Maxiprep DNA):

5.2 ug vector of interest plasmid4.35 ug AAV 1 serotype plasmid4.35 ug AAV 2 serotype plasmid10.4 ug pDF6 plasmid (adenovirus helper genes) □ Vortex to mixAdd 434 uL DMEM (no serum!)Add 130 ul PEI solutionVortex 5-10 secondsAdd DNA/DMEM/PEI mixture to prewarmed mediaVortex briefly to mixReplace media in 15 cm dish with DNA/DMEM/PEI mixtureReturn to 37° C. incubatorIncubate 48h before harvesting (make sure medium isn't turning tooacidic)

Virus Harvest:

1. aspirate media carefully from 15 cm dish dishes (advantageously donot dislodge cells)2. Add 25 ml RT DPBS (Invitrogen) to each plate and gently remove cellswith a cell scraper. Collect suspension in 50 ml tubes.3. Pellet cells at 800×g for 10 minutes.4. Discard supernatant

Pause Point: Freeze Cell Pellet at −80C if Desired

5. resuspend pellet in 150 mM NaCl, 20 mM Tris pH 8.0, use 10 ml pertissue culture plate.6. Prepare a fresh solution of 10% sodium deoxycholate in dH2O. Add 1.25ml of this per tissue culture plate for a final concentration of 0.5%.Add benzonase nuclease to a final concentration of 50 units per ml. Mixtube thoroughly.7. Incubate at 37° C. for 1 hour (Waterbath).8. Remove cellular debris by centrifuging at 3000×g for 15 mins.Transfer to fresh 50 ml tube and ensure all cell debris has been removedto prevent blocking of heparin columns.

Heparin Column Purification of AAV1/2:

1. Set up HiTrap heparin columns using a peristaltic pump so thatsolutions flow through the column at 1 ml per minute. It is important toensure no air bubbles are introduced into the heparin column.2. Equilibrate the column with 10 ml 150 mM NaCl, 20 mM Tris, pH 8.0using the peristaltic pump.3. Binding of virus: Apply 50 ml virus solution to column and allow toflow through.4. Wash step 1: column with 20 ml 100 mM NaCl, 20 mM Tris, pH 8.0.(using the peristaltic pump)5. Wash step 2: Using a 3 ml or 5 ml syringe continue to wash the columnwith 1 ml 200 mM NaCl, 20 mM Tris, pH 8.0, followed by 1 ml 300 mM NaCl,20 mM Tris, pH 8.0.Discard the flow-through.(prepare the syringes with different buffers during the 50 min flowthrough of virus solution above)6. Elution Using 5 ml syringes and gentle pressure (flow rate of<lml/min) elute the virus from the column by applying:

1.5 ml 400 mM NaCl, 20 mM Tris, pH 8.0 3.0 ml 450 mM NaCl, 20 mM Tris,pH 8.0 1.5 ml 500 mM NaCl, 20 mM Tris, pH 8.0

Collect these in a 15 ml centrifuge tube.

Concentration of AAV1/2:

1. Concentration step 1: Concentrate the eluted virus using Amicon ultra15 ml centrifugal filter units with a 100,000 molecular weight cutoff.Load column eluate into the concentrator and centrifuge at 2000×g for 2minutes (at room temperature. Check concentrated volume—it should beapproximately 500 μl. If necessary, centrifuge in 1 min intervals untilcorrect volume is reached.2. buffer exchange: Add 1 ml sterile DPBS to filter unit, centrifuge in1 min intervals until correct volume (500 ul) is reached.3. Concentration step 2: Add 500 ul concentrate to an Amicon Ultra 0.5ml 100K filter unit. Centrifuge at 6000 g for 2 min. Check concentratedvolume—it should be approximately 100 μl. If necessary, centrifuge in 1min intervals until correct volume is reached.4. Recovery: Invert filter insert and insert into fresh collection tube.Centrifuge at 1000 g for 2 min.Aliquot and freeze at −80° C.1 ul is typically required per injection site, small aliquots (e.g. 5ul) are therefore recommended (avoid freeze-thaw of virus). determineDNaseI-resistant GC particle titer using qPCR (see separate protocol)

Materials

Amicon Ultra, 0.5 ml, 100K; MILLIPORE; UFC510024 Amicon Ultra, 15 ml,100K; MILLIPORE; UFC910024

Benzonase nuclease; Sigma-Aldrich, E1014HiTrap Heparin cartridge; Sigma-Aldrich; 54836Sodium deoxycholate; Sigma-Aldrich; D5670

AAV1 Supernatant Production Protocol

Media: D10+HEPES

500 ml bottle DMEM high glucose+Glutamax (Invitrogen)50 ml Hyclone FBS (heat-inactivated) (Thermo Fischer)5.5 ml HEPES solution (1M, GIBCO)Cells: low passage HEK293FT (passage <10 at time of virus production)Thaw new cells of passage 2-4 for virus production, grow up for 2-5passagesTransfection reagent: Polyethylenimine (PEI) “Max”Dissolve 50 mg PEI “Max” in 50 ml sterile Ultrapure H₂O

Adjust pH to 7.1

Filter with 0.22 um fliptop filterSeal tube and wrap with parafilmFreeze aliquots at −20° C. (for storage, can also be used immediately)

Cell Culture

Culture low passage HEK293FT in D10+HEPES Passage everyday between 1:2and 1:2.5Advantageously do let cells reach more than 85% confluency

For T75

Warm 10 ml HBSS (—Mg2+, —Ca2+, GIBCO)+1 ml TrypLE Express (GIBCO) perflask to 37° C. (Waterbath)

Aspirate media fully

Add 10 ml warm HBSS gently (to wash out media completely)

Add 1 ml TrypLE per Flask

Place flask in incubator (37° C.) for 1 min

Rock flask to detach cells

Add 9 ml D10+HEPES media (37° C.)

Pipette up and down 5 times to generate single cell suspension

Split at 1:2-1:2.5 (12 ml media for T75) ratio (if cells are growingmore slowly, discard and thaw a new batch, they are not in optimalgrowth)

transfer to T225 as soon as enough cells are present (for ease ofhandling large amounts of cells)

AAV production (single 15 cm dish scale)Plate 10 million cells in 21.5 ml media into a 15 cm dishIncubate for 18-22 hours at 37° C.Transfection is ideal at 80% confluence per platePrewarm 22 ml media (D10+HEPES)Prepare tube with DNA mixture (use endofree maxiprep DNA):5.2 ug vector of interest plasmid8.7 ug AAV 1 serotype plasmid10.4 ug DF6 plasmid (adenovirus helper genes)

Vortex to mix

Add 434 uL DMEM (no serum!) Add 130 ul PEI solutionVortex 5-10 secondsAdd DNA/DMEM/PEI mixture to prewarmed mediaVortex briefly to mixReplace media in 15 cm dish with DNA/DMEM/PEI mixtureReturn to 37° C. incubatorIncubate 48h before harvesting (advantageously monitor to ensure mediumis not turning too acidic)

Virus harvest:

Remove supernatant from 15 cm dishFilter with 0.45 um filter (low protein binding) Aliquot and freeze at−80° C.Transduction (primary neuron cultures in 24-well format, 5 DIV)Replace complete neurobasal media in each well of neurons to betransduced with freshneurobasal (usually 400 ul out of 500 ul per well is replaced)Thaw AAV supernatant in 37° C. waterbathLet equilibrate in incubator for 30 minAdd 250 ul AAV supernatant to each well

Incubate 24h at 37° C.

Remove media/supernatant and replace with fresh complete neurobasalExpression starts to be visible after 48h, saturates around 6-7 DaysPost InfectionConstructs for pAAV plasmid with GOI should not exceed 4.8 kb includingboth ITRS.

Example of a human codon optimized sequence (i.e. being optimized forexpression in humans) sequence: SaCas9 is provided below:

(SEQ ID NO: 196) ACCGGTGCCACCATGTACCCATACGATGTTCCAGATTACGCTTCGCCGAAGAAAAAGCGCAAGGTCGAAGCGTCCATGAAAAGGAACTACATTCTGGGGCTGGACATCGGGATTACAAGCGTGGGGTATGGGATTATTGACTATGAAACAAGGGACGTGATCGACGCAGGCGTCAGACTGTTCAAGGAGGCCAACGTGGAAAACAATGAGGGACGGAGAAGCAAGAGGGGAGCCAGGCGCCTGAAACGACGGAGAAGGCACAGAATCCAGAGGGTGAAGAAACTGCTGTTCGATTACAACCTGCTGACCGACCATTCTGAGCTGAGTGGAATTAATCCTTATGAAGCCAGGGTGAAAGGCCTGAGTCAGAAGCTGTCAGAGGAAGAGTTTTCCGCAGCTCTGCTGCACCTGGCTAAGCGCCGAGGAGTGCATAACGTCAATGAGGTGGAAGAGGACACCGGCAACGAGCTGTCTACAAAGGAACAGATCTCACGCAATAGCAAAGCTCTGGAAGAGAAGTATGTCGCAGAGCTGCAGCTGGAACGGCTGAAGAAAGATGGCGAGGTGAGAGGGTCAATTAATAGGTTCAAGACAAGCGACTACGTCAAAGAAGCCAAGCAGCTGCTGAAAGTGCAGAAGGCTTACCACCAGCTGGATCAGAGCTTCATCGATACTTATATCGACCTGCTGGAGACTCGGAGAACCTACTATGAGGGACCAGGAGAAGGGAGCCCCTTCGGATGGAAAGACATCAAGGAATGGTACGAGATGCTGATGGGACATTGCACCTATTTTCCAGAAGAGCTGAGAAGCGTCAAGTACGCTTATAACGCAGATCTGTACAACGCCCTGAATGACCTGAACAACCTGGTCATCACCAGGGATGAAAACGAGAAACTGGAATACTATGAGAAGTTCCAGATCATCGAAAACGTGTTTAAGCAGAAGAAAAAGCCTACACTGAAACAGATTGCTAAGGAGATCCTGGTCAACGAAGAGGACATCAAGGGCTACCGGGTGACAAGCACTGGAAAACCAGAGTTCACCAATCTGAAAGTGTATCACGATATTAAGGACATCACAGCACGGAAAGAAATCATTGAGAACGCCGAACTGCTGGATCAGATTGCTAAGATCCTGACTATCTACCAGAGCTCCGAGGACATCCAGGAAGAGCTGACTAACCTGAACAGCGAGCTGACCCAGGAAGAGATCGAACAGATTAGTAATCTGAAGGGGTACACCGGAACACACAACCTGTCCCTGAAAGCTATCAATCTGATTCTGGATGAGCTGTGGCATACAAACGACAATCAGATTGCAATCTTTAACCGGCTGAAGCTGGTCCCAAAAAAGGTGGACCTGAGTCAGCAGAAAGAGATCCCAACCACACTGGTGGACGATTTCATTCTGTCACCCGTGGTCAAGCGGAGCTTCATCCAGAGCATCAAAGTGATCAACGCCATCATCAAGAAGTACGGCCTGCCCAATGATATCATTATCGAGCTGGCTAGGGAGAAGAACAGCAAGGACGCACAGAAGATGATCAATGAGATGCAGAAACGAAACCGGCAGACCAATGAACGCATTGAAGAGATTATCCGAACTACCGGGAAAGAGAACGCAAAGTACCTGATTGAAAAAATCAAGCTGCACGATATGCAGGAGGGAAAGTGTCTGTATTCTCTGGAGGCCATCCCCCTGGAGGACCTGCTGAACAATCCATTCAACTACGAGGTCGATCATATTATCCCCAGAAGCGTGTCCTTCGACAATTCCTTTAACAACAAGGTGCTGGTCAAGCAGGAAGAGAACTCTAAAAAGGGCAATAGGACTCCTTTCCAGTACCTGTCTAGTTCAGATTCCAAGATCTCTTACGAAACCTTTAAAAAGCACATTCTGAATCTGGCCAAAGGAAAGGGCCGCATCAGCAAGACCAAAAAGGAGTACCTGCTGGAAGAGCGGGACATCAACAGATTCTCCGTCCAGAAGGATTTTATTAACCGGAATCTGGTGGACACAAGATACGCTACTCGCGGCCTGATGAATCTGCTGCGATCCTATTTCCGGGTGAACAATCTGGATGTGAAAGTCAAGTCCATCAACGGCGGGTTCACATCTTTTCTGAGGCGCAAATGGAAGTTTAAAAAGGAGCGCAACAAAGGGTACAAGCACCATGCCGAAGATGCTCTGATTATCGCAAATGCCGACTTCATCTTTAAGGAGTGGAAAAAGCTGGACAAAGCCAAGAAAGTGATGGAGAACCAGATGTTCGAAGAGAAGCAGGCCGAATCTATGCCCGAAATCGAGACAGAACAGGAGTACAAGGAGATTTTCATCACTCCTCACCAGATCAAGCATATCAAGGATTTCAAGGACTACAAGTACTCTCACCGGGTGGATAAAAAGCCCAACAGAGAGCTGATCAATGACACCCTGTATAGTACAAGAAAAGACGATAAGGGGAATACCCTGATTGTGAACAATCTGAACGGACTGTACGACAAAGATAATGACAAGCTGAAAAAGCTGATCAACAAAAGTCCCGAGAAGCTGCTGATGTACCACCATGATCCTCAGACATATCAGAAACTGAAGCTGATTATGGAGCAGTACGGCGACGAGAAGAACCCACTGTATAAGTACTATGAAGAGACTGGGAACTACCTGACCAAGTATAGCAAAAAGGATAATGGCCCCGTGATCAAGAAGATCAAGTACTATGGGAACAAGCTGAATGCCCATCTGGACATCACAGACGATTACCCTAACAGTCGCAACAAGGTGGTCAAGCTGTCACTGAAGCCATACAGATTCGATGTCTATCTGGACAACGGCGTGTATAAATTTGTGACTGTCAAGAATCTGGATGTCATCAAAAAGGAGAACTACTATGAAGTGAATAGCAAGTGCTACGAAGAGGCTAAAAAGCTGAAAAAGATTAGCAACCAGGCAGAGTTCATCGCCTCCTTTTACAACAACGACCTGATTAAGATCAATGGCGAACTGTATAGGGTCATCGGGGTGAACAATGATCTGCTGAACCGCATTGAAGTGAATATGATTGACATCACTTACCGAGAGTATCTGGAAAACATGAATGATAAGCGCCCCCCTCGAATTATCAAAACAATTGCCTCTAAGACTCAGAGTATCAAAAAGTACTCAACCGACATTCTGGGAAACCTGTATGAGGTGAAGAGCAAAAAGCACCCTCAGATTATCAAAAAGGGCTAAGAATTC

Example 30 Minimizing Off-Target Cleavage Using Cas9 Nickase and TwoGuide RNAs

Cas9 is a RNA-guided DNA nuclease that may be targeted to specificlocations in the genome with the help of a 20 bp RNA guide. However theguide sequence may tolerate some mismatches between the guide sequenceand the DNA-target sequence. The flexibility is undesirable due to thepotential for off-target cleavage, when the guide RNA targets Cas9 to aan off-target sequence that has a few bases different from the guidesequence. For all experimental applications (gene targeting, cropengineering, therapeutic applications, etc) it is important to be ableto improve the specificity of Cas9 mediated gene targeting and reducethe likelihood of off-target modification by Cas9.

Applicants developed a method of using a Cas9 nickase mutant incombination with two guide RNAs to facilitate targeted double strandbreaks in the genome without off-target modifications. The Cas9 nickasemutant may be generated from a Cas9 nuclease by disabling its cleavageactivity so that instead of both strands of the DNA duplex being cleavedonly one strand is cleaved. The Cas9 nickase may be generated byinducing mutations in one ore more domains of the Cas9 nuclease, e.g.Ruvc1 or HNH. These mutations may include but are not limited tomutations in a Cas9 catalytic domain, e.g. in SpCas9 these mutations maybe at positions D10 or H840. These mutations may include but are notlimited to D10A, E762A, H840A, N854A, N863A or D986A in SpCas9 butnickases may be generated by inducing mutations at correspondingpositions in other CRISPR enzymes or Cas9 orthologs. In a most preferredembodiment of the invention the Cas9 nickase mutant is a SpCas9 nickasewith a D10A mutation.

The way this works is that each guide RNA in combination with Cas9nickase would induce the targeted single strand break of a duplex DNAtarget. Since each guide RNA nicks one strand, the net result is adouble strand break. The reason this method eliminates off-targetmutations is because it is very unlikely to have an off-target site thathas high degrees of similarity for both guide sequences (20 bp+2bp(PAM)=22 bp specificity for each guide, and two guides means anyoff-target site will have to have close to 44 bp of homologoussequence). Although it is still likely that individual guides may haveoff-targets, but those off-targets will only be nicked, which isunlikely to be repaired by the mutagenic NHEJ process. Therefore themultiplexing of DNA double strand nicking provides a powerful way ofintroducing targeted DNA double strand breaks without off-targetmutagenic effects.

Applicants carried out experiments involving the co-transfection ofHEK293FT cells with a plasmid encoding Cas9 (D10A) nickase as well asDNA expression cassettes for one or more guides. Applicants transfectedcells using Lipofectamine 2000, and transfected cells were harvested 48or 72 hours after transfections. Double nicking-induced NHEJ weredetected using the SURVEYOR nuclease assay as described previouslyherein (FIGS. 33, 34 and 35).

Applicants have further identified parameters that relate to efficientcleavage by the Cas9 nickase mutant when combined with two guide RNAsand these parameters include but are not limited to the length of the 5′overhang. Efficient cleavage is reported for 5′ overhang of at least 26base pairs. In a preferred embodiment of the invention, the 5′ overhangis at least 30 base pairs and more preferably at least 34 base pairs.Overhangs of up to 200 base pairs may be acceptable for cleavage, while5′ overhangs less than 100 base pairs are preferred and 5′ overhangsless than 50 base pairs are most preferred (FIGS. 36 and 37).

Example 31 Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced GenomeEditing Specificity

Targeted genome editing technologies have enabled a broad range ofresearch and medical applications. The Cas9 nuclease from the microbialCRISPR-Cas system is targeted to specific genomic loci by a 20-nt guidesequence, which can tolerate certain mismatches to the DNA target andthereby promote undesired off-target mutagenesis. As briefly describedin Example 30, in this example Applicants further describe an approachthat combines a Cas9 nickase mutant with paired guide RNAs to introducetargeted double-strand breaks. Since individual nicks in the genome arerepaired with high fidelity, simultaneous nicking via appropriatelyoffset guide RNAs is required for double stranded breaks and thuseffectively extends the number of specifically recognized bases fortarget cleavage. Applicants demonstrated that using paired nicking canreduce off-target activity by 50-1,000 fold in cell lines and facilitategene knockout in mouse zygotes without sacrificing on-target cleavageefficiency. This versatile strategy thus enables a wide variety ofgenome editing applications that require high specificity.

The ability to perturb the genome in a precise and targeted fashion iscrucial for understanding genetic contributions to biology and disease.Genome engineering of cell lines or animal models has traditionally beenaccomplished through random mutagenesis or low-efficiency genetargeting. To facilitate genome editing, programmable sequence-specificDNA nuclease technologies have enabled targeted modification ofendogenous genomic sequences with high efficiency, particularly inspecies that have proven genetically intractable. The RNA-guided Cas9nucleases from the microbial CRISPR (clustered regularly interspacedshort palindromic repeat)-Cas systems are robust and versatile tools forstimulating targeted double-stranded DNA breaks (DSBs) in eukaryoticcells, where the resulting cellular repair mechanisms—non-homologous endjoining (NHEJ) or homology-directed repair (HDR) pathways—can beexploited to induce error-prone or defined alterations.

The Cas9 nuclease from Streptococcus pyogenes can be directed by achimeric single guide RNA (sgRNA) to any genomic locus preceding a5′-NGG protospacer-adjacent motif (PAM). A 20-nt guide sequence withinthe sgRNA directs Cas9 to the genomic target via Watson-Crick basepairing and can be easily programmed to target a desired genomic locus.Recent studies of Cas9 specificity have demonstrated that although eachbase within the 20-nt guide sequence contributes to overall specificity,multiple mismatches between the guide RNA and its complementary targetDNA sequence can be tolerated depending on the quantity, position, andbase identity of mismatches, leading to potential off-target DSBs andindel formation. These unwanted mutations can potentially limit theutility of Cas9 for genome editing applications that require high levelsof precision, such as generation of isogenic cell lines for testingcausal genetic variations or in vivo and ex vivo genome editing-basedtherapies.

To improve the specificity of Cas9-mediated genome editing, Applicantsdeveloped a novel strategy that combines the D10A mutant nickase versionof Cas9 (Cas9n) with a pair of offset sgRNAs complementary to oppositestrands of the target site. While nicking of both DNA strands by a pairof Cas9 nickases leads to site-specific DSBs and NHEJ, individual nicksare predominantly repaired by the high-fidelity base excision repairpathway (BER). A paired nickase strategy which may relate to thepossibility for engineering a system to ameliorate off-target activitymay be referred to in the Mali et al., 2013a paper titled CAS9transcriptional activators for target specificity screening and pairednickases for cooperative genome engineering. In a manner analogous todimeric zinc finger nucleases (ZFNs) and transcription activator-likeeffector nucleases (TALENs), where DNA cleavage requires synergisticinteraction of two independent specificity-encoding DNA-binding modulesdirecting FokI nuclease monomers, this double nicking strategy minimizesoff-target mutagenesis by each individual Cas9n-sgRNA complex whilemaintaining on-target modification rates similar to those of wild typeCas9. Here Applicants define crucial parameters for the selection ofsgRNA pairs that facilitate effective double nicking, compare thespecificity of wildtype Cas9 and Cas9n with double nicking, anddemonstrate a variety of experimental applications that can be achievedusing double nicking in cells as well as in mouse zygotes.

Extension of guide sequence does not improve Cas9 targeting specificity:Cas9 targeting is facilitated by base-pairing between the 20-nt guidesequence within the sgRNA and the target DNA. Applicants reasoned thatcleavage specificity might be improved by increasing the length ofbase-pairing between the guide RNA and its target locus. Applicantsgenerated U6-driven expression cassettes to express three sgRNAs with20-nt (sgRNA 1) or 30-nt guide sequences (sgRNAs 2 and 3) targeting alocus within the human EMX1 gene (FIG. 38A).

Applicants and others have previously shown that while single-basemismatches between the PAM-distal region of the guide sequence andtarget DNA are well-tolerated by Cas9, multiple mismatches in thisregion can significantly affect on-target activity. To determine whetheradditional PAM-distal bases (21-30) could influence overall targetingspecificity, Applicants designed sgRNAs 2 and 3 to contain 10 additionalbases consisting of either 10 perfectly matched or 8 mismatched bases(bases 21-28). Surprisingly, Applicants observed that these extendedsgRNAs mediated similar levels of modification at the target locus inHEK 293FT cells regardless of whether the additional bases werecomplementary to the genomic target (FIG. 38B). Subsequent Northernblots revealed that the majority of both sgRNA 2 and 3 were processed tothe same length as sgRNA 1, which contains the same 20-nt guide sequencewithout additional bases (FIG. 38C).

Cas9 nickase generates efficient NHEJ with paired, offset guide RNAs:Given that extension of the guide sequence failed to improve Cas9targeting specificity, Applicants sought an alternative strategy forincreasing the overall base-pairing length between the guide sequenceand its DNA target. Cas9 enzymes contain two conserved nuclease domains,HNH and RuvC, which cleave the DNA strand complementary andnon-complementary to the guide RNA, respectively. Mutations of thecatalytic residues (D10A in RuvC and H840A in HNH) convert Cas9 into DNAnickases. As single-strand nicks are preferentially repaired by thehigh-fidelity BER pathway, Applicants reasoned that two Cas9 nickingenzymes directed by a pair of sgRNAs targeting opposite strands of atarget locus could mediate DSBs while minimizing off-target activity(FIG. 39A).

A number of factors may affect cooperative nicking leading to indelformation, including steric hindrance between two adjacent Cas9molecules or Cas9-sgRNA complexes, overhang type, and sequence context;some of these may be characterized by testing multiple sgRNA pairs withdistinct target sequences and offsets (the distance between thePAM-distal (5′) ends of the guide sequence of a given sgRNA pair). Tosystematically assess how sgRNA offsets might affect subsequent repairand generation of indels, Applicants first designed sets of sgRNA pairstargeted against the human EMX1 genomic locus separated by a range ofoffset distances from approximately −200 to 200 bp to create both 5′-and 3′-overhang products (FIG. 39A, FIG. 44). Applicants then assessedthe ability of each sgRNA pair, with the D10A Cas9 mutant (referred toas Cas9n; H840A Cas9 mutant is referred to as Cas9H840A), to generateindels in human HEK 293FT cells. Robust NHEJ (up to 40%) was observedfor sgRNA pairs with offsets from −4 to 20 bp, with modest indelsforming in pairs offset by up to 100-bp (FIG. 39B, left panel).Applicants subsequently recapitulated these findings by testingsimilarly offset sgRNA pairs at two other genomic loci, DYRK1A andGRIN2B (FIG. 39B, right panels). Notably, across all three lociexamined, only sgRNA pairs creating 5′ overhangs with less than 8 bpoverlap between the guide sequences (offset greater than −8 bp) wereable to mediate detectable indel formation (FIG. 39C). Importantly, eachguide used in these assays is able to efficiently induce indels whenpaired with wildtype Cas9 (FIG. 44), indicating that the relativepositions of the guide pairs are the most important parameters inpredicting double nicking activity.

Since Cas9n and Cas9H840A nick opposite strands of DNA, substitution ofCas9n with Cas9H840A with a given sgRNA pair should result in theinversion of the overhang type. For example, a pair of sgRNAs that willgenerate a 5′ overhang with Cas9n should in principle generate thecorresponding 3′ overhang instead. Therefore, sgRNA pairs that lead tothe generation of a 3′ overhang with Cas9n might be used with Cas9H840Ato generate a 5′ overhang. Unexpectedly, Applicants tested Cas9H840Awith a set of sgRNA pairs designed to generate both 5′ and 3′ overhangs(offset range from −278 to +58 bp), but were unable to observe indelformation. Further work may be needed to identify the necessary designrules for sgRNA pairing to allow double nicking by Cas9H840A.

Double nicking mediates efficient genome editing with improvedspecificity: Having established that double nicking (DN) mediates highefficiency NHEJ at levels comparable to those induced by wildtype Cas9,Applicants next studied whether DN has improved specificity overwildtype Cas9 by measuring their off-target activities. Applicantsco-delivered Cas9n with sgRNAs 1 and 9, spaced by a +23 bp offset, totarget the human EMX1 locus in HEK 293FT cells (FIG. 40A). This DNconfiguration generated on-target indel levels similar to thosegenerated by the wildtype Cas9 paired with each sgRNA alone (FIG. 40B,left panel). Strikingly, unlike with wildtype Cas9, DN did not generatedetectable modification at a previously validated sgRNA 1 off-targetsite, OT-4, by SURVEYOR assay (FIG. 40B, right panel), suggesting thatDN can potentially reduce the likelihood of off-target modifications.

Using deep sequencing to assess modification at 5 different sgRNA 1off-target loci (FIG. 40A), Applicants observed significant mutagenesisat all sites with wild type Cas9+sgRNA 1 (FIG. 40C). In contrast,cleavage by Cas9n at 5 off-target sites tested was barely detectableabove background sequencing error. Using the ratio of on- to off-targetmodification levels as a metric of specificity, Applicants found thatCas9n with a pair of sgRNAs was able to achieve over 100-fold greaterspecificity relative to wild type Cas9 with one of the sgRNAs (FIG.40D). Applicants conducted additional off-target analysis by deepsequencing for two sgRNA pairs (offsets of +16 and +20 bp) targeting theVEGFA locus, with similar results (FIG. 40E). DN at these off-targetloci (Table 4) was able to achieve 200 to over 1500-fold greaterspecificity than the wild-type Cas9 (FIG. 40F, FIG. 44). Taken together,these results demonstrate that Cas9-mediated double nicking minimizesoff-target mutagenesis and is suitable for genome editing with increasedspecificity.

Double nicking facilitates high-efficiency homology directed repair,NHEJ-mediated DNA insertion, and genomic microdeletions: DSBs canstimulate homology directed repair (HDR) to enable highly preciseediting of genomic target sites. To evaluate DN-induced HDR, Applicantstargeted the human EMX1 locus with pairs of sgRNAs offset by −3 and +18bp (generating 31- and 52-bp 5′ overhangs), respectively, and introduceda single-stranded oligodeoxynucleotide (ssODN) bearing a HindIIIrestriction site as the HDR repair template (FIG. 41A). Each DN sgRNApair successfully induced HDR at frequencies higher than those ofsingle-guide Cas9n nickases and comparable to those of wild-type Cas9(FIG. 41B). Furthermore, genome editing in embryonic stem cells orpatient derived induced pluripotent stem cells represents a keyopportunity for generating and studying new disease paradigms as well asdeveloping new therapeutics. Since single nick approaches to inducingHDR in human embryonic stem cells (hESCs) have met with limited success,Applicants attempted DN in the HUES62 hES cell line and observedsuccessful HDR (FIG. 41C).

To further characterize how offset sgRNA spacing affects the efficiencyof HDR, Applicants next tested in HEK 293FT cells a set of sgRNA pairswhere the cleavage site of at least one sgRNA is situated near the siteof recombination (overlapping with the HDR ssODN donor template arm).Applicants observed that sgRNA pairs generating 5′ overhangs and havingat least one nick occurring within 22 bp of the homology arm are able toinduce HDR at levels comparable to those of wildtype Cas9-mediated HDR,and significantly greater than those of single Cas9n-sgRNA nicking. Incontrast, Applicants did not observe HDR with sgRNA pairs that generated3′-overhangs or double nicking of the same DNA strand (FIG. 41D).

The ability to create defined overhangs could enable precise insertionof donor repair templates containing compatible overhangs viaNHEJ-mediated ligation. To explore this alternative strategy fortransgene insertion, Applicants targeted the EMX1 locus with Cas9n andan sgRNA pair designed to generate a 43 bp 5′-overhang near the stopcodon, and supplied a double-stranded oligonucleotide (dsODN) duplexwith matching overhangs (FIG. 42A). The annealed dsODN insert,containing multiple epitope tags and a restriction site, wassuccessfully integrated into the target at a frequency of 3% (1/37screened by Sanger sequencing of cloned amplicons). This ligation-basedstrategy thus illustrates an effective approach for inserting dsODNsencoding short modifications such as protein tags or recombination sitesinto an endogenous locus.

Additionally, Applicants targeted combinations of sgRNA pairs (4 sgRNAsper combination) to the DYRK1A locus in HEK 293FT cells to facilitategenomic microdeletions. Applicants generated a set of sgRNAs to mediate0.5 kb, 1 kb, 2 kb, and 6 kb deletions (FIG. 42B, FIG. 45: sgRNAs 32,33, 54-61) and verified successful multiplex nicking-mediated deletionover these ranges via PCR screen of predicted deletion sizes.

Double nicking enables efficient genome modification in mouse zygotes:Recent work demonstrated that co-delivery of wildtype Cas9 mRNA alongwith multiple sgRNAs can mediate single-step generation of transgenicmice carrying multiple allelic modifications. Given the ability toachieve genome modification in vivo using several sgRNAs at once,Applicants sought to assess the efficiency of multiple nicking by Cas9nin mouse zygotes. Cytoplasmic co-injection of wildtype Cas9 or Cas9nmRNA and sgRNAs into single-cell mouse zygotes allowed successfultargeting of the Mecp2 locus (FIG. 43A). To identify the optimalconcentration of Cas9n mRNA and sgRNA for efficient gene targeting,Applicants titrated Cas9 mRNA from 100 ng/uL to 3 ng/uL whilemaintaining the sgRNA levels at a 1:20 Cas9:sgRNA molar ratio. Allconcentrations tested for Cas9 double nicking mediated modifications inat least 80% of embryos screened, similar to levels achieved by wildtypeCas9 (FIG. 43B). Taken together, these results suggest a number ofapplications for double nicking-based genome editing.

Discussion: Given the permanent nature of genomic modifications,specificity is of paramount importance to sensitive applications such asstudies aimed at linking specific genetic variants with biologicalprocesses or disease phenotypes and gene therapy. Applicants haveexplored strategies to improve the targeting specificity of Cas9.Although simply extending the guide sequence length of sgRNA failed toimprove targeting specificity, combining two appropriately offset sgRNAswith Cas9n effectively generated indels while minimizing unwantedcleavage since individual off-target single-stranded nicks are repairedwith high fidelity via base excision repair. Given that significantoff-target mutagenesis has been previously reported for Cas9 nucleasesin human cells, the DN approach could provide a generalizable solutionfor rapid and accurate genome editing. The characterization of spacingparameters governing successful

Cas9 double nickase-mediated gene targeting reveals an effective offsetwindow over 100-bp long, allowing for a high degree of flexibility inthe selection of sgRNA pairs. Previous computational analyses haverevealed an average targeting range of every 12-bp for the Streptococcuspyogenes Cas9 in the human genome based on the 5′-NGG PAM, which suggestthat appropriate sgRNA pairs should be readily identifiable for mostloci within the genome. Applicants have additionally demonstratedDN-mediated indel frequencies comparable to wild type Cas9 modificationat multiple genes and loci in both human and mouse cells, confirming thereproducibility of this strategy for high-precision genome engineering(FIG. 44).

The Cas9 double nicking approach is in principle similar to ZFN andTALEN-based genome editing systems, where cooperation between twohemi-nuclease domains is required to achieve double-stranded break atthe target site. Systematic studies of ZFN and TALEN systems haverevealed that the targeting specificity of a given ZFN and TALEN paircan be highly dependent on the nuclease architecture (homo- orheterodimeric nucleases) or target sequence, and in some cases TALENscan be highly specific. Although the wildtype Cas9 system has been shownto exhibit high levels of off-target mutagenesis, the DN system is apromising solution and brings RNA-guided genome editing to similarspecificity levels as ZFNs and TALENs.

Additionally, the ease and efficiency with which Cas9 can be targetedrenders the DN system especially attractive. However, DNA targetingusing DN will likely face similar off-target challenges as ZFNs andTALENs, where cooperative nicking at off-target sites might still occur,albeit at a significantly reduced likelihood. Given the extensivecharacterization of Cas9 specificity and sgRNA mutation analysis, aswell as the NHEJ-mediating sgRNA offset range identified in this study,computational approaches may be used to evaluate the likely off-targetsites for a given pair of sgRNAs. To facilitate sgRNA pair selection,Applicants developed an online web tool that identifies sgRNAcombinations with optimal spacing for double nicking applications(available at the website genome-engineering.org).

Although Cas9n has been previously shown to facilitate HDR at on-targetsites, its efficiency is substantially lower than that of wildtype Cas9.The double nicking strategy, by comparison, maintains high on-targetefficiencies while reducing off-target modifications to backgroundlevels. Nevertheless, further characterizations of DN off-targetactivity, particularly via whole genome sequencing and targeted deepsequencing of cells or whole organisms generated using the DN approach,may be needed to evaluate the utility of Cas9n DN in biotechnological orclinical applications that require ultra-high precision genome editing.Additionally, Cas9n has been shown to induce low levels of indels aton-target sites for certain sgRNAs as indicated in the Mali et al. paperentitled “CAS9 transcriptional activators for target specificityscreening and paired nickases for cooperative genome engineering”(2013), which may result from residual double-strand break activitiesand be circumvented by further structure-function studies of Cas9catalytic activity. Overall, Cas9n-mediated multiplex nicking serves asa customizable platform for highly precise and efficient targeted genomeengineering and promises to broaden the range of applications inbiotechnology, basic science, and medicine.

Experimental Procedures

Cell culture and transfection: Human embryonic kidney (HEK) cell line293FT (Life Technologies) or mouse Neuro 2a (Sigma-Aldrich) cell linewas maintained in Dulbecco's modified Eagle's Medium (DMEM) supplementedwith 10% fetal bovine serum (HyClone), 2 mM GlutaMAX (LifeTechnologies), 100 U/mL penicillin, and 100 m/mL streptomycin at 37° C.with 5% CO2 incubation.

Cells were seeded onto 24-well plates (Corning) at a density of 120,000cells/well, 24 hours prior to transfection. Cells were transfected usingLipofectamine 2000 (Life Technologies) at 80-90% confluency followingthe manufacturer's recommended protocol. A total of 500 ng Cas9 plasmidand 100 ng of U6-sgRNA PCR product was transfected.

Human embryonic stem cell line HUES62 (Harvard Stem Cell Institute core)was maintained in feeder-free conditions on GelTrex (Life Technologies)in mTesR medium (Stemcell Technologies) supplemented with 100 ug/mlNormocin (InvivoGen). HUES62 cells were transfected with Amaxa P3Primary Cell 4-D Nucleofector Kit (Lonza) following the manufacturer'sprotocol.

SURVEYOR nuclease assay for genome modification: 293FT and HUES62 cellswere transfected with DNA as described above. Cells were incubated at37° C. for 72 hours post-transfection prior to genomic DNA extraction.Genomic DNA was extracted using the QuickExtract DNA Extraction Solution(Epicentre) following the manufacturer's protocol. Briefly, pelletedcells were resuspended in QuickExtract solution and incubated at 65° C.for 15 minutes, 68° C. for 15 minutes, and 98° C. for 10 minutes.

The genomic region flanking the CRISPR target site for each gene was PCRamplified (Table 2), and products were purified using QiaQuick SpinColumn (Qiagen) following the manufacturer's protocol. 400 ng total ofthe purified PCR products were mixed with 2 μl 10×Taq DNA Polymerase PCRbuffer (Enzymatics) and ultrapure water to a final volume of 20 μl, andsubjected to a re-annealing process to enable heteroduplex formation:95° C. for 10 min, 95° C. to 85° C. ramping at −2° C./s, 85° C. to 25°C. at −0.25° C./s, and 25° C. hold for 1 minute. After re-annealing,products were treated with SURVEYOR nuclease and SURVEYOR enhancer S(Transgenomics) following the manufacturer's recommended protocol, andanalyzed on 4-20% Novex TBE poly-acrylamide gels (Life Technologies).Gels were stained with SYBR Gold DNA stain (Life Technologies) for 30minutes and imaged with a Gel Doc gel imaging system (Bio-rad).Quantification was based on relative band intensities. Indel percentagewas determined by the formula, 100×(1−(1−(b+c)/(a+b+c))½), where a isthe integrated intensity of the undigested PCR product, and b and c arethe integrated intensities of each cleavage product.

Northern blot analysis of tracrRNA expression in human cells Northernblots were performed as previously described in Cong et al., 2013.Briefly, RNAs were extracted using the mirPremier microRNA Isolation Kit(Sigma) and heated to 95° C. for 5 min before loading on 8% denaturingpolyacrylamide gels (SequaGel, National Diagnostics). Afterwards, RNAwas transferred to a pre-hybridized Hybond N+ membrane (GE Healthcare)and crosslinked with Stratagene UV Crosslinker (Stratagene). Probes werelabeled with [gamma-32P] ATP (Perkin Elmer) with T4 polynucleotidekinase (New England Biolabs). After washing, membrane was exposed tophosphor screen for one hour and scanned with phosphorimager (Typhoon).

Deep sequencing to assess targeting specificity: HEK 293FT cells wereplated and transfected as described above, 72 hours prior to genomic DNAextraction. The genomic region flanking the CRISPR target site for eachgene was amplified (see Table 3 for primer sequences) by a fusion PCRmethod to attach the Illumina P5 adapters as well as uniquesample-specific barcodes to the target. PCR products were purified usingEconoSpin 96-well Filter Plates (Epoch Life Sciences) following themanufacturer's recommended protocol.

Barcoded and purified DNA samples were quantified by Qubit 2.0Fluorometer (Life Technologies) and pooled in an equimolar ratio.Sequencing libraries were then sequenced with the Illumina MiSeqPersonal Sequencer (Life Technologies).

Sequencing data analysis, indel detection, and homologous recombinationdetection: MiSeq reads were filtered by requiring an average Phredquality (Q score) of at least 30, as well as perfect sequence matches tobarcodes and amplicon forward primers. Reads from on- and off-targetloci were analyzed by performing Ratcliff-Obershelp string comparison,as implemented in the Python difflib module, against loci sequences thatincluded 30 nucleotides upstream and downstream of the target site (atotal of 80 bp). The resulting edit operations were parsed, and readswere counted as indels if insertion or deletion operations were found.Analyzed target regions were discarded if part of their alignment felloutside the MiSeq read itself or if more than 5 bases were uncalled.

Negative controls for each sample provided a gauge for the inclusion orexclusion of indels as putative cutting events. For quantification ofhomologous recombination, reads were first processed as in the indeldetection workflow, and then checked for presence of homologousrecombination template CCAGGCTTGG (SEQ ID NO: 197).

Microinjection into mouse zygotes: Cas9 mRNA and sgRNA templates wereamplified with T7 promoter sequence-conjugated primers. After gelpurification, Cas9 and Cas9n were transcribed with mMESSAGE mMACHINE T7Ultra Kit (Life technologies). sgRNAs were transcribed withMEGAshortscript T7 Kit (Life technologies). RNAs were purified byMEGAclear Kit (Life technologies) and frozen at −80° C.

MII-stage oocytes were collected from 8-week old superovulated BDF 1females by injecting 7.5 I.U. of PMSG (Harbor, UCLA) and hCG(Millipore). They were transferred into HTF medium supplemented with 10mg/ml bovine serum albumin (BSA; Sigma-Aldrich) and inseminated withcapacitated sperm obtained from the caudal epididymides of adult C57BL/6male mice. Six hours after fertilization, zygotes were injected withmRNAs and sgRNAs in M2 media (Millipore) using a Piezo impact-drivenmicromanipulator (Prime Tech Ltd., Ibaraki, Japan). The concentrationsof Cas9 and Cas9n mRNAs and sgRNAs are described in the text and FIG.6B. After microinjection, zygotes were cultured in KSOM (Millipore) in ahumidified atmosphere of 5% CO2 and 95% air at 37° C.

Genome extraction from blastocyst embryos: Following in vitro culture ofembryos for 6 days, the expanded blastocysts were washed with 0.01% BSAin PBS and individually collected into 0.2 mL tubes. Five microliters ofgenome extraction solution (50 mM Tris-HCl, pH 8.0, 0.5% Triton-X100, 1mg/ml Proteinase K) were added and the samples were incubated in 65° C.for 3 hours followed by 95° C. for 10 min. Samples were then amplifiedfor targeted deep sequencing as described above.

TABLE 2 Primers used for SURVEYOR assays. Related to ExperimentalProcedures. genomic SEQ ID primer name target primer sequence (5′ to 3′)NO: SUV901 EMX1 CCATCCCCTTCTGTGAATGT 180 SUV902 EMX1GGAGATTGGAGACACGGAGA 181 DYRK1A-F DYRK1A GGAGCTGGTCTGTTGGAGAA 198DYRK1A-R DYRK1A TCCCAATCCATAATCCCACGTT 199 GRIN2B-F GRIN2BCAGGAGGGCCAGGAGATTTG 200 GRIN2B-R GRIN2B TGAAATCGAGGATCTGGGCG 201 F1VEGFA CAAAGGACCCCAGTCACTCC 202 R1 VEGFA GAGGAGGGAGCAGGAAAGTG 203 F2VEGFA GACACTTCCCAAAGGACCCC 204 R2 VEGFA TGAGAGCCGTTCCCTCTTTG 205 F3VEGFA GACAGGGGCAAAGTGAGTGA 206 R3 VEGFA TTCATGGTTTCGGAGGCCC 207 F4 VEGFATGAGTGACCTGCTTTTGGGG 208 R4 VEGFA GTTCATGGTTTCGGAGGCCC 209

TABLE 3 Primers used to generate amplicons for next- generationsequencing. Related to Experimental Procedures. SEQ ID primer nameprimer sequence (5′ to 3′) NO: EMX1-F GGAGGACAAAGTACAAACGGC 210 EMX1-RATCGATGTCCTCCCCATTGG 211 EMX1-HR-F CCATCCCCTTCTGTGAATGT 180 EMX1-HR-RGGAGATTGGAGACACGGAGA 181 EMX1-OT1.1-F TGGGAGAGAGACCCCTTCTT 212EMX1-OT1.1-R TCCTGCTCTCACTTAGACTTTCTC 213 EMX1-OT1.2-FGACATTCCTCCTGAGGGAAAA 214 EMX1-OT1.2-R GATAAAATGTATTCCTTCTCACCATTC 215EMX1-OT1.3-F CCAGACTCAGTAAAGCCTGGA 216 EMX1-OT1.3-R TGGCCCCAGTCTCTCTTCTA217 EMX1-OT1.4-F CACGGCCTTTGCAAATAGAG 218 EMX1-OT1.4-RCATGACTTGGCCTTTGTAGGA 219 EMX1-OT1.5-F TGGGGTTACAGAAAGAATAGGG 220EMX1-OT1.5-R TTCTGAGGGCTGCTACCTGT 221 VEGFA-F TGAAGCAACTCCAGTCCCAA 222VEGFA-R CCCGGCTCTGGCTAAAGAG 223 VEGFA-OT96.1-F TGGGTGTGCACATCTAAGGA 224VEGFA-OT96.1-R CCACTGAGTCAACTGTAAGCA 225 VEGFA-OT98.1-FGCAGAGGAATATGTGACATGAGG 226 VEGFA-OT98.1-R TACTCCCTGCTGTCCTCTCC 227VEGFA-OT98.2-F TTCCTGGCCAAGTCGATTCC 228 VEGFA-OT98.2-RGGATACCAGCATGGGCTACC 229 VEGFA-OT98.3-F ACTCTTGAGATTGGAACGGGA 230VEGFA-OT98.3-R CCACACTTATCTACGCCCCA 231 VEGFA-OT598.4-FAGCCAGAAGAGAACATCCACG 232 VEGFA-OT98.4-R AGTTGCTCTTTGTTGAGAGGGA 233MECP2-F TGACCGGGGACCTATGTATGA 234 MECP2-R ACAAGACTTGCTCTTACTTACTTGA 235

TABLE 4 VEGFA off-target site sequences. Related to ExperimentalProcedures. Off-target Off-target SEQ ID sgRNA site sequence (5′ to 3′)NO: 96 OT-96.1 GGATGGAGGGAGTTTGCTCCTGG 236 98 OT-98.1CGCCCTCCCCACCCCGCCTCCGG 237 98 OT-98.2 TACCCCCCACACCCCGCCTCTGG 238 98OT-98.3 GGGCCCCTCCACCCCGCCTCTGG 239 98 OT-98.4 CTACCCCTCCACCCCGCCTCCGG240

TABLE 5 Primers used to screen for multiplex nicking inducedmicrodeletion in the EMX1 locus. Related to Experimental Procedures.Deletion size Forward Reverse (kb) primer (5′ to 3′) primer (5′ to 3′)0.5 AGGTTGTTGCTGTTGCTTTAC AGCACGGTTAATTTGCATACA A (SEQ ID NO: 241) TCT(SEQ ID NO: 243) 1 AGGTTGTTGCTGTTGCTTTAC TCCAGGCAGTTTTCTTCTGGT A (SEQ IDNO: 241) (SEQ ID NO: 244) 2 AGGTTGTTGCTGTTGCTTTAC CCAGAGAGCCAGCATTCCAA A(SEQ ID NO: 241) (SEQ ID NO: 245) 6 AGGTTTCACCTGGTTTGGGGAGGGCTCCCACTAGAAGAGG (SEQ ID NO: 242) (SEQ ID NO: 246)

All sequences are in the 5′ to 3′ direction. For U6 transcription, thestring of underlined Ts serve as the transcriptional terminator.

> sgRNA containing +85 tracrRNA (Streptococcus pyogenes SF370) (SEQ IDNO: 247)gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccNNNNNNNNNNNNNNNNNNNN 

 gaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcTTTTTTT (guide sequence is the bold stretch of upper case “N”s and thetracrRNA fragment is the sequence in bold before the string ofunderlined Ts) > 3xFLAG-NLS-SpCas9-NLS (SEQ ID NO: 248)ATGGACTATAAGGACCACGACGGAGACTACAAGGATCATGATATTGATTACAAAGACGATGACGATAAGATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGCAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACAAGCGTCCTGCTGCTACTAAGAAAGCTGGTCAAGCTAAGAAAAAGAAA > 3xFLAG-NLS-SpCas9n-NLS (the D10A nickasemutation is highlighted in bold) (SEQ ID NO: 249)ATGGACTATAAGGACCACGACGGAGACTACAAGGATCATGATATTGATTACAAAGACGATGACGATAAGATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGACAAGAAGTACAGCATCGGCCTGGCCATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGCAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACAAGCGTCCTGCTGCTACTAAGAAAGCTGGTCAAGCTAAGAAAAAGAAA T7-SpCas9-NLS (T7 promoter is underlined) (SEQID NO: 250) GCCACCGGTTAATACGACTCACTATAGGGCCACCATGGACTATAAGGACCACGACGGAGACTACAAGGATCATGATATTGATTACAAAGACGATGACGATAAGATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAAACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGCAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGTAAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACC T7-SpCas9n-NLS (D10A nickasemutation is highlighted in bold; T7 promoter is underlined) (SEQ ID NO:251) GCCACCGGTTAATACGACTCACTATAGGGCCACCATGGACTATAAGGACCACGACGGAGACTACAAGGATCATGATATTGATTACAAAGACGATGACGATAAGATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGACAAGAAGTACAGCATCGGCCTGGCCATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGCAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGTAAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACC > dsODN ligation insert fwd(SEQ ID NO: 252)AGCAGGCCAATGGGGAGGACATCGATGTCACCTCCAATGACTAcTACCCATACGATGTTCCAGATTACGCTATGGACTATAAGGACCACGACGGAGACTACAAGGATCATGATATTGATTACAAAGACGATGACGATAAGaagcttgaaATGGCATCAATGCAGAAGCTGATCTCAGAGGAGGACCTGtaa > dsODN ligation insert rev (SEQ ID NO: 253)TAGTCATTGGAGGTGACATCGATGTCCTCCCCATTGGCCTGCTTTACAGGTCCTCCTCTGAGATCAGCTTCTGCATTGATGCCATTTCAAGCTTCTTATCGTCATCGTCTTTGTAATCAATATCATGATCCTTGTAGTCTCCGTCGTGGTCCTTATAGTCCATAGCGTAATCTGGAACATCGTATGGGTAG

Example 32 Further Characterization of Double Nicking by RNA-GuidedCRISPR Cas9

Double nicking specificity: The targeting space for Cas9 multiplexednicking can be increased by exploiting a relaxation in the stringency ofCas9 PAM recognition when the target DNA is targeted by multiple guideRNAs (sgRNAs). Applciants have demonstrated in Example 31 (Ran et al.,Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editingspecificity, Cell. 2013 Sep. 12; 154(6):1380-9) that two appropriatelyoffset sgRNAs can be targeted within a specific sgRNA offset′ parameterto generate indels. Applicants demonstrate that efficient indels can beformed when offset sgRNAs are targeted with non-NGG PAMs (in particularNGN and NNG). It appears that Cas9 can potentially tolerate a shiftedPAM or a PAM with only one G nucleotide in the 2nd or 3rd PAM basepositions.

Cas9 mutagenesis for double nicking: Applicants tested the ability ofCas9 nickase mutants to generate indels when targeted by only one sgRNA.Single-stranded DNA breaks are repaired through high fidelity, non-NHEJrepair pathways. However, work by other groups may suggest significantmutagenesis by the Cas9(D10A) nickase and one sgRNA (Mali et al.,Science 2013). Applicants generated mutations in the two additional (of3 total) RuvC catalytic domains present within S. pyogenes Cas9, makinga Cas9 3KO [Cas9(D10A, E762A, and D986A)] and tested it against theAAVS1 locus (sgRNA guide sequence: ggggccactagggacaggat (SEQ ID NO:254)). Applicants found lower indel mutagenesis by the Cas9 3K0 vs. theD10A nickase mutant (see table below in which Data is average of 3biological replicates.)

Average Standard error indels of mean D10A 0.0267 0.008854089 3KO 0.00670.003340519 wtCas9 9.5927 0.394065483

Specific editing of mouse embryos: Applicants titrated Cas9 dosage inmouse zygotes from 10 ng/uL to 0.01 ng/uL and determined the optimaldosage for specific Cas9 editing by double nicking. At the 10 ng/uLcondition, Applicants demonstrated 88% editing with wild-type Cas9 and83% editing by D10A double nicking. D10A+L sgRNA at 10 ng/uL exhibited0% indels while wt Cas9 exhibited no indels at the 1, 0.1, and 0.01ng/uL condition.

The experimental details for this example with regard to cell cultureand transfection protocols and SURVEYOR nuclease assays for genomemodification are further described in Example 31.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention.

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What is claimed is:
 1. A method of modifying an organism or a non-humanorganism by minimizing off-target modifications by manipulation of afirst and a second target sequence on opposite strands of a DNA duplexin a genomic locus of interest in a cell comprising delivering anon-naturally occurring or engineered composition comprising: I. a firstCRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence, whereinthe first polynucleotide sequence comprises: (a) a first guide sequencecapable of hybridizing to the first target sequence, (b) a first tracrmate sequence, and (c) a first tracr sequence, II. a second CRISPR-Cassystem chiRNA polynucleotide sequence, wherein the second polynucleotidesequence comprises: (a) a second guide sequence capable of hybridizingto the second target sequence, (b) a second tracr mate sequence, and (c)a second tracr sequence, and III. a polynucleotide sequence encoding aCRISPR enzyme comprising at least one or more nuclear localizationsequences and comprising one or more mutations, wherein (a), (b) and (c)are arranged in a 5′ to 3′ orientation, wherein when transcribed, thefirst and the second tracr mate sequence hybridize to the first andsecond tracr sequence respectively and the first and the second guidesequence directs sequence-specific binding of a first and a secondCRISPR complex to the first and second target sequences respectively,wherein the first CRISPR complex comprises the CRISPR enzyme complexedwith (1) the first guide sequence that is hybridizable to the firsttarget sequence, and (2) the first tracr mate sequence that ishybridized to the first tracr sequence, wherein the second CRISPRcomplex comprises the CRISPR enzyme complexed with (1) the second guidesequence that is hybridizable to the second target sequence, and (2) thesecond tracr mate sequence that is hybridized to the second tracrsequence, wherein the polynucleotide sequence encoding a CRISPR enzymeis DNA or RNA, and wherein the first guide sequence directs cleavage ofone strand of the DNA duplex near the first target sequence and thesecond guide sequence directs cleavage of other strand near the secondtarget sequence inducing a double strand break, thereby modifying theorganism or the non-human organism by minimizing off-targetmodifications.
 2. The method of claim 1, wherein the first guidesequence directing cleavage of one strand of the DNA duplex near thefirst target sequence and the second guide sequence directing cleavageof other strand near the second target sequence results in a 5′overhang.
 3. The method of claim 2, wherein the 5′ overhang is at most200 base pairs.
 4. The method of claim 2, wherein the 5′ overhang is atmost 100 base pairs.
 5. The method of claim 2, wherein the 5′ overhangis at most 50 base pairs.
 6. The method of claim 2, wherein the 5′overhang is at least 26 base pairs.
 7. The method of claim 2, whereinthe 5′ overhang is at least 30 base pairs.
 8. The method of claim 2,wherein the 5′ overhang is 34-50 base pairs.
 9. The method of claim 2,wherein the 5′ overhang is at least 15 base pairs.
 10. The method ofclaim 2, wherein the 5′ overhang is at least 10 base pairs.
 11. Themethod of claim 2, wherein the 5′ overhang is at least 1 base pair. 12.The method of claim 2, wherein the 5′ overhang is 1-34 base pairs. 13.The method of claim 1, wherein the first guide sequence directingcleavage of one strand of the DNA duplex near the first target sequenceand the second guide sequence directing cleavage of other strand nearthe second target sequence results in a blunt cut.
 14. The method ofclaim 1, wherein the first guide sequence directing cleavage of onestrand of the DNA duplex near the first target sequence and the secondguide sequence directing cleavage of other strand near the second targetsequence results in a 3′ overhang.
 15. The method of claim 14, whereinthe 3′ overhang is at most 150 base pairs.
 16. The method of claim 14,wherein the 3′ overhang is at most 100 base pairs.
 17. The method ofclaim 14, wherein the 3′ overhang is at most 50 base pairs.
 18. Themethod of claim 14, wherein the 3′ overhang is at most 25 base pairs.19. The method of claim 14, wherein the 3′ overhang is at least 15 basepairs.
 20. The method of claim 14, wherein the 3′ overhang is at least10 base pairs.
 21. The method of claim 14, wherein the 3′ overhang is atleast 1 base pair.
 22. The method of claim 14, wherein the 3′ overhangis 1-100 base pairs.
 23. The method of claim 1, wherein any or all ofthe polynucleotide sequence encoding the CRISPR enzyme, the first andthe second guide sequence, the first and the second tracr mate sequenceor the first and the second tracr sequence, is/are RNA.
 24. The methodof claim 1, wherein the polynucleotides comprising the sequence encodingthe CRISPR enzyme, the first and the second guide sequence, the firstand the second tracr mate sequence or the first and the second tracrsequence, is/are RNA and are delivered via nanoparticles, exosomes,microvesicles, or a gene-gun.
 25. The method of claim 1, wherein thefirst and second tracr mate sequence share 100% identity.
 26. The methodof claim 1, wherein the first and second tracr sequence share 100%identity.
 27. The method of claim 1, wherein the CRISPR enzyme is a Cas9enzyme.
 28. The method of claim 27, wherein the CRISPR enzyme is SpCas9.29. The method of claim 28, wherein the CRISPR enzyme comprises one ormore mutations in a catalytic domain.
 30. The method of claim 29,wherein the one or more mutations is in a RuvC domain.
 31. The method ofclaim 30, wherein the one or more mutations is selected from the groupconsisting of D10A, E762A and D986A.
 32. The method of claim 31, whereinthe CRISPR enzyme has the D10A mutation.
 33. The method of claim 29,wherein the one or more mutations is in a HNH domain.
 34. The method ofclaim 33, wherein the one or more mutations is selected from the groupconsisting of H840A, N854A and N863A.
 35. The method of claim 34,wherein the CRISPR enzyme has the H840A mutation.
 36. A method ofmodifying an organism or a non-human organism by minimizing off-targetmodifications by manipulation of a first and a second target sequence onopposite strands of a DNA duplex in a genomic locus of interest in acell comprising delivering a non-naturally occurring or engineeredcomposition comprising a vector system comprising one or more vectorscomprising I. a first regulatory element operably linked to (a) a firstguide sequence capable of hybridizing to the first target sequence, and(b) at least one or more tracr mate sequences, II. a second regulatoryelement operably linked to (a) a second guide sequence capable ofhybridizing to the second target sequence, and (b) at least one or moretracr mate sequences, III. a third regulatory element operably linked toan enzyme-coding sequence encoding a CRISPR enzyme, and IV. a fourthregulatory element operably linked to a tracr sequence, whereincomponents I, II, III and IV are located on the same or differentvectors of the system, when transcribed, the tracr mate sequencehybridizes to the tracr sequence and the first and the second guidesequence directs sequence-specific binding of a first and a secondCRISPR complex to the first and second target sequences respectively,wherein the first CRISPR complex comprises the CRISPR enzyme complexedwith (1) the first guide sequence that is hybridizable to the firsttarget sequence, and (2) the tracr mate sequence that is hybridized tothe tracr sequence, wherein the second CRISPR complex comprises theCRISPR enzyme complexed with (1) the second guide sequence that ishybridizable to the second target sequence, and (2) the tracr matesequence that is hybridized to the tracr sequence, wherein thepolynucleotide sequence encoding a CRISPR enzyme is DNA or RNA, andwherein the first guide sequence directs cleavage of one strand of theDNA duplex near the first target sequence and the second guide sequencedirects cleavage of other strand near the second target sequenceinducing a double strand break, thereby modifying the organism or thenon-human organism by minimizing off-target modifications.
 37. Themethod of claim 36, wherein the first guide sequence directing cleavageof one strand of the DNA duplex near the first target sequence and thesecond guide sequence directing cleavage of other strand near the secondtarget sequence results in a 5′ overhang.
 38. The method of claim 37,wherein the 5′ overhang is at most 200 base pairs.
 39. The method ofclaim 37, wherein the 5′ overhang is at most 100 base pairs.
 40. Themethod of claim 37, wherein the 5′ overhang is at most 50 base pairs.41. The method of claim 37, wherein the 5′ overhang is at least 26 basepairs.
 42. The method of claim 37, wherein the 5′ overhang is at least30 base pairs.
 43. The method of claim 37, wherein the 5′ overhang is34-50 base pairs.
 44. The method of claim 37, wherein the 5′ overhang isat least 15 base pairs.
 45. The method of claim 37, wherein the 5′overhang is at least 10 base pairs.
 46. The method of claim 37, whereinthe 5′ overhang is at least 1 base pair.
 47. The method of claim 37,wherein the 5′ overhang is 1-34 base pairs.
 48. The method of claim 36,wherein the first guide sequence directing cleavage of one strand of theDNA duplex near the first target sequence and the second guide sequencedirecting cleavage of other strand near the second target sequenceresults in a blunt cut.
 49. The method of claim 36, wherein the firstguide sequence directing cleavage of one strand of the DNA duplex nearthe first target sequence and the second guide sequence directingcleavage of other strand near the second target sequence results in a 3′overhang.
 50. The method of claim 49, wherein the 3′ overhang is at most150 base pairs.
 51. The method of claim 49, wherein the 3′ overhang isat most 100 base pairs.
 52. The method of claim 49, wherein the 3′overhang is at most 50 base pairs.
 53. The method of claim 49, whereinthe 3′ overhang is at most 25 base pairs.
 54. The method of claim 49,wherein the 3′ overhang is at least 15 base pairs.
 55. The method ofclaim 49, wherein the 3′ overhang is at least 10 base pairs.
 56. Themethod of claim 49, wherein the 3′ overhang is at least 1 base pair. 57.The method of claim 49, wherein the 3′ overhang is 1-100 base pairs. 58.The method of claim 36, wherein any or all of the polynucleotidesequence encoding the CRISPR enzyme, the first and the second guidesequence, the first and the second tracr mate sequence or the first andthe second tracr sequence, is/are RNA.
 59. The method of claim 36,wherein the first and second tracr mate sequence share 100% identity.60. The method of claim 36, wherein the first and second tracr sequenceshare 100% identity.
 61. The method of claim 36, wherein the CRISPRenzyme is a Cas9 enzyme.
 62. The method of claim 61, wherein the CRISPRenzyme is SpCas9.
 63. The method of claim 62, wherein the CRISPR enzymecomprises one or more mutations in a catalytic domain.
 64. The method ofclaim 63, wherein the one or more mutations is in a RuvC domain.
 65. Themethod of claim 64, wherein the one or more mutations is selected fromthe group consisting of D10A, E762A and D986A.
 66. The method of claim65, wherein the CRISPR enzyme has the D10A mutation.
 67. The method ofclaim 63, wherein the one or more mutations is in a HNH domain.
 68. Themethod of claim 67, wherein the one or more mutations is selected fromthe group consisting of H840A, N854A and N863A.
 69. The method of claim68, wherein the CRISPR enzyme has the H840A mutation.
 70. The method ofclaim 36, wherein one or more of the viral vectors are delivered viananoparticles, exosomes, microvesicles, or a gene-gun.
 71. A method ofmodifying a genomic locus of interest by minimizing off-targetmodifications by introducing into a cell containing and expressing adouble stranded DNA molecule encoding the gene product an engineered,non-naturally occurring CRISPR-Cas system comprising a Cas proteinhaving one or more mutations and two guide RNAs that target a firststrand and a second strand of the DNA molecule respectively, whereby theguide RNAs target the DNA molecule encoding the gene product and the Casprotein nicks each of the first strand and the second strand of the DNAmolecule encoding the gene product, whereby expression of the geneproduct is altered; and, wherein the Cas protein and the two guide RNAsdo not naturally occur together.
 72. The method of claim 71, wherein theCas protein nicking each of the first strand and the second strand ofthe DNA molecule encoding the gene product results in a 5′ overhang. 73.The method of claim 71, wherein the 5′ overhang is at most 200 basepairs.
 74. The method of claim 71, wherein the 5′ overhang is at most100 base pairs.
 75. The method of claim 71, wherein the 5′ overhang isat most 50 base pairs.
 76. The method of claim 71, wherein the 5′overhang is at least 26 base pairs.
 77. The method of claim 71, whereinthe 5′ overhang is at least 30 base pairs.
 78. The method of claim 71,wherein the 5′ overhang is 34-50 base pairs.
 79. The method of claim 71,wherein the 5′ overhang is at least 15 base pairs.
 80. The method ofclaim 71, wherein the 5′ overhang is at least 10 base pairs.
 81. Themethod of claim 71, wherein the 5′ overhang is at least 1 base pair. 82.The method of claim 71, wherein the 5′ overhang is 1-34 base pairs. 83.The method of claim 71, wherein the Cas protein nicking each of thefirst strand and the second strand of the DNA molecule encoding the geneproduct results in a blunt cut.
 84. The method of claim 71, wherein theCas protein nicking each of the first strand and the second strand ofthe DNA molecule encoding the gene product results in a 3′ overhang. 85.The method of claim 84, wherein the 3′ overhang is at most 150 basepairs.
 86. The method of claim 84, wherein the 3′ overhang is at most100 base pairs.
 87. The method of claim 84, wherein the 3′ overhang isat most 50 base pairs.
 88. The method of claim 84, wherein the 3′overhang is at most 25 base pairs.
 89. The method of claim 84, whereinthe 3′ overhang is at least 15 base pairs.
 90. The method of claim 84,wherein the 3′ overhang is at least 10 base pairs.
 91. The method ofclaim 84, wherein the 3′ overhang is at least 1 base pair.
 92. Themethod of claim 84, wherein the 3′ overhang is 1-100 base pairs.
 93. Themethod of claim 71, wherein the guide RNAs comprise a guide sequencefused to a tracr mate sequence and a tracr sequence.
 94. The method ofclaim 71, wherein the Cas protein is codon optimized for expression in aeukaryotic cell.
 95. The method of claim 94, wherein the eukaryotic cellis a mammalian cell.
 96. The method of claim 95, wherein the mammaliancell is a human cell.
 97. The method of claim 71, wherein the Casprotein is a type II CRISPR-Cas protein.
 98. The method of claim 97,wherein the Cas protein is a Cas9 protein.
 99. The method of claim 98,wherein the Cas protein is SpCas9.
 100. The method of claim 99, whereinthe Cas protein comprises one or more mutations in a catalytic domain.101. The method of claim 100, wherein the one or more mutations is in aRuvC domain.
 102. The method of claim 101, wherein the one or moremutations is selected from the group consisting of D10A, E762A andD986A.
 103. The method of claim 102, wherein the Cas protein has theD10A mutation.
 104. The method of claim 100, wherein the one or moremutations is in a HNH domain.
 105. The method of claim 104, wherein theone or more mutations is selected from the group consisting of H840A,N854A and N863A.
 106. The method of claim 105, wherein the Cas proteinhas the H840A mutation.
 107. The method of claim 71, wherein theexpression of the gene product is decreased.
 108. The method of claim71, wherein a template polynucleotide is further introduced into the DNAmolecule encoding the gene product or an intervening sequence is excisedprecisely allowing 5′ overhangs to reanneal and ligate.
 109. The methodof claim 71, wherein the expression of the gene product is increased orthe activity or function of the gene product is altered.
 110. The methodof claim 71, wherein the gene product is a protein.
 111. An engineered,non-naturally occurring CRISPR-Cas system comprising a Cas proteinhaving one or more mutations and two guide RNAs that target a firststrand and a second strand respectively of a double stranded DNAmolecule encoding a gene product in a cell, whereby the guide RNAstarget the DNA molecule encoding the gene product and the Cas proteinnicks each of the first strand and the second strand of the DNA moleculeencoding the gene product, whereby expression of the gene product isaltered; and, wherein the Cas protein and the two guide RNAs do notnaturally occur together.
 112. The CRISPR-Cas system of claim 111,wherein the guide RNAs comprise a guide sequence fused to a tracr matesequence and a tracr sequence.
 113. The CRISPR-Cas system of claim 111,wherein the Cas protein is a type II CRISPR-Cas protein.
 114. TheCRISPR-Cas system of claim 113, wherein the Cas protein is a Cas9protein.
 115. The CRISPR-Cas system of claim 114, wherein the Casprotein is SpCas9.
 116. The CRISPR-Cas system of claim 115, wherein theCas protein comprises one or more mutations in a catalytic domain. 117.The CRISPR-Cas system of claim 116, wherein the one or more mutations isin a RuvC domain.
 118. The CRISPR-Cas system of claim 117, wherein theone or more mutations is selected from the group consisting of D10A,E762A and D986A.
 119. The CRISPR-Cas system of claim 118, wherein theCas protein has the D10A mutation.
 120. The CRISPR-Cas system of claim116, wherein the one or more mutations is in a HNH domain.
 121. TheCRISPR-Cas system of claim 120, wherein the one or more mutations isselected from the group consisting of H840A, N854A and N863A.
 122. TheCRISPR-Cas system of claim 121, wherein the Cas protein has the H840Amutation.
 123. The CRISPR-Cas system of claim 111, wherein the Casprotein is codon optimized for expression in a eukaryotic cell.
 124. TheCRISPR-Cas system of claim 123, wherein the eukaryotic cell is amammalian cell.
 125. The CRISPR-Cas system of claim 124, wherein themammalian cell is a human cell.
 126. The CRISPR-Cas system of claim 111,wherein the expression of the gene product is decreased.
 127. TheCRISPR-Cas system of claim 111, wherein a template polynucleotide isfurther introduced into the DNA molecule encoding the gene product or anintervening sequence is excised precisely allowing 5′ or 3′ overhangs toreanneal and ligate.
 128. The CRISPR-Cas system of claim 111, whereinthe expression of the gene product is increased or the activity orfunction of the gene product is altered.
 129. The CRISPR-Cas system ofclaim 111, wherein the gene product is a protein.
 130. An engineered,non-naturally occurring vector system comprising one or more vectorscomprising: a) a first regulatory element operably linked to each of twoCRISPR-Cas system guide RNAs that target a first strand and a secondstrand respectively of a double stranded DNA molecule encoding a geneproduct, b) a second regulatory element operably linked to a Casprotein, wherein components (a) and (b) are located on same or differentvectors of the system, whereby the guide RNAs target the DNA moleculeencoding the gene product and the Cas protein nicks each of the firststrand and the second strand of the DNA molecule encoding the geneproduct, whereby expression of the gene product is altered; and, whereinthe Cas protein and the two guide RNAs do not naturally occur together.131. The vector system of claim 130, wherein the guide RNAs comprise aguide sequence fused to a tracr mate sequence and a tracr sequence. 132.The vector system of claim 130, wherein the Cas protein is a type IICRISPR-Cas protein.
 133. The vector system of claim 132, wherein the Casprotein is a Cas9 protein.
 134. The vector system of claim 133, whereinthe Cas protein is SpCas9.
 135. The vector system of claim 134, whereinthe Cas protein comprises one or more mutations in a catalytic domain.136. The vector system of claim 135, wherein the one or more mutationsis in a RuvC domain.
 137. The vector system of claim 136, wherein theone or more mutations is selected from the group consisting of D10A,E762A and D986A.
 138. The vector system of claim 137, wherein the Casprotein has the D10A mutation.
 139. The vector system of claim 135,wherein the one or more mutations is in a HNH domain.
 140. The vectorsystem of claim 139, wherein the one or more mutations is selected fromthe group consisting of H840A, N854A and N863A.
 141. The vector systemof claim 140, wherein the Cas protein has the H840A mutation.
 142. Thevector system of claim 130, wherein the Cas protein is codon optimizedfor expression in a eukaryotic cell.
 143. The vector system of claim142, wherein the eukaryotic cell is a mammalian cell.
 144. The vectorsystem of claim 143, wherein the mammalian cell is a human cell. 145.The vector system of claim 130, wherein the gene product is a protein.146. The vector system of claim 130, wherein the expression of the geneproduct is decreased.
 147. The vector system of claim 130, wherein atemplate polynucleotide is further introduced into the DNA moleculeencoding the gene product or an intervening sequence is excisedprecisely allowing 5′ or 3′ overhangs to reanneal and ligate.
 148. Thevector system of claim 130, wherein the expression of the gene productis increased or the activity or function of the gene product is altered.149. The vector system of claim 130, wherein the vectors of the systemare viral vectors.
 150. The vector system of claim 130, wherein thevectors of the system are delivered via nanoparticles, exosomes,microvesicles, or a gene-gun.
 151. A method of modifying an organismcomprising a first and a second target sequence on opposite strands of aDNA duplex in a genomic locus of interest in a cell by promotinghomology directed repair comprising delivering a non-naturally occurringor engineered composition comprising: I. a first CRISPR-Cas systemchimeric RNA (chiRNA) polynucleotide sequence, wherein the firstpolynucleotide sequence comprises: (a) a first guide sequence capable ofhybridizing to the first target sequence, (b) a first tracr matesequence, and (c) a first tracr sequence, II. a second CRISPR-Cas systemchiRNA polynucleotide sequence, wherein the second polynucleotidesequence comprises: (a) a second guide sequence capable of hybridizingto the second target sequence, (b) a second tracr mate sequence, and (c)a second tracr sequence, and III. a polynucleotide sequence encoding aCRISPR enzyme comprising at least one or more nuclear localizationsequences and comprising one or more mutations, IV. a repair templatecomprising a synthesized or engineered single-stranded oligonucleotide,wherein (a), (b) and (c) are arranged in a 5′ to 3′ orientation, whereinwhen transcribed, the first and the second tracr mate sequence hybridizeto the first and second tracr sequence respectively and the first andthe second guide sequence directs sequence-specific binding of a firstand a second CRISPR complex to the first and second target sequencesrespectively, wherein the first CRISPR complex comprises the CRISPRenzyme complexed with (1) the first guide sequence that is hybridizableto the first target sequence, and (2) the first tracr mate sequence thatis hybridized to the first tracr sequence, wherein the second CRISPRcomplex comprises the CRISPR enzyme complexed with (1) the second guidesequence that is hybridizable to the second target sequence, and (2) thesecond tracr mate sequence that is hybridized to the second tracrsequence, wherein the polynucleotide sequence encoding a CRISPR enzymeis DNA or RNA, wherein the first guide sequence directs cleavage of onestrand of the DNA duplex near the first target sequence and the secondguide sequence directs cleavage of other strand near the second targetsequence inducing a double strand break, and wherein the repair templateis introduced into the DNA duplex by homologous recombination, wherebythe organism is modified.
 152. The method of claim 151 wherein therepair template further comprises a restriction endonuclease restrictionsite.
 153. The method of claim 151, wherein the first guide sequencedirecting cleavage of one strand of the DNA duplex near the first targetsequence and the second guide sequence directing cleavage of otherstrand near the second target sequence results in a 5′ or 3′ overhang.154. The method of claim 153, wherein the 5′ overhang is 1-200 basepairs.
 155. The method of claim 153, wherein the 3′ overhang is 1-100base pairs.
 156. The method of claim 151, wherein any or all of thepolynucleotide sequence encoding the CRISPR enzyme, the first and thesecond guide sequence, the first and the second tracr mate sequence orthe first and the second tracr sequence, is/are RNA.
 157. The method ofclaim 151, wherein the polynucleotides comprising the sequence encodingthe CRISPR enzyme, the first and the second guide sequence, the firstand the second tracr mate sequence or the first and the second tracrsequence, is/are RNA and are delivered via nanoparticles, exosomes,microvesicles, or a gene-gun.
 158. The method of claim 151, wherein thefirst and second tracr mate sequence share 100% identity.
 159. Themethod of claim 151, wherein the first and second tracr sequence share100% identity.
 160. The method of claim 151, wherein the CRISPR enzymeis a Cas9 enzyme.
 161. The method of claim 160, wherein the CRISPRenzyme is SpCas9.
 162. The method of claim 161, wherein the CRISPRenzyme comprises one or more mutations in a catalytic domain.
 163. Themethod of claim 162, wherein the one or more mutations is in a RuvCdomain.
 164. The method of claim 163, wherein the one or more mutationsis selected from the group consisting of D10A, E762A and D986A.
 165. Themethod of claim 164, wherein the CRISPR enzyme has the D10A mutation.166. The method of claim 162, wherein the one or more mutations is in aHNH domain.
 167. The method of claim 166, wherein the one or moremutations is selected from the group consisting of H840A, N854A andN863A.
 168. The method of claim 167, wherein the CRISPR enzyme has theH840A mutation.
 169. A method of modifying an organism comprising afirst and a second target sequence on opposite strands of a DNA duplexin a genomic locus of interest in a cell by facilitating non homologousend joining (NHEJ) mediated ligation comprising delivering anon-naturally occurring or engineered composition comprising: I. a firstCRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence, whereinthe first polynucleotide sequence comprises: (a) a first guide sequencecapable of hybridizing to the first target sequence, (b) a first tracrmate sequence, and (c) a first tracr sequence, II. a second CRISPR-Cassystem chiRNA polynucleotide sequence, wherein the second polynucleotidesequence comprises: (a) a second guide sequence capable of hybridizingto the second target sequence, (b) a second tracr mate sequence, and (c)a second tracr sequence, and III. a polynucleotide sequence encoding aCRISPR enzyme comprising at least one or more nuclear localizationsequences and comprising one or more mutations, IV. a repair templatecomprising a first set of overhangs, wherein (a), (b) and (c) arearranged in a 5′ to 3′ orientation, wherein when transcribed, the firstand the second tracr mate sequence hybridize to the first and secondtracr sequence respectively and the first and the second guide sequencedirects sequence-specific binding of a first and a second CRISPR complexto the first and second target sequences respectively, wherein the firstCRISPR complex comprises the CRISPR enzyme complexed with (1) the firstguide sequence that is hybridizable to the first target sequence, and(2) the first tracr mate sequence that is hybridized to the first tracrsequence, wherein the second CRISPR complex comprises the CRISPR enzymecomplexed with (1) the second guide sequence that is hybridizable to thesecond target sequence, and (2) the second tracr mate sequence that ishybridized to the second tracr sequence, wherein the polynucleotidesequence encoding a CRISPR enzyme is DNA or RNA, wherein the first guidesequence directs cleavage of one strand of the DNA duplex near the firsttarget sequence and the second guide sequence directs cleavage of otherstrand near the second target sequence inducing a double strand breakwith a second set of overhangs, wherein the first set of overhangs iscompatible with and matches the second set of overhangs, and wherein therepair template is introduced into the DNA duplex by ligation, wherebythe organism is modified.
 170. The method according to claim 169 whereinthe repair template is a synthesized or engineered double-strandedoligonucleotide duplex.
 171. The method according to claim 169 whereinthe repair template is generated from a piece of DNA that is introducedinto the cell and is enzymatically processed.
 172. The method of claim169, wherein the first guide sequence directing cleavage of one strandof the DNA duplex near the first target sequence and the second guidesequence directing cleavage of other strand near the second targetsequence results in a 5′ or 3′ overhang.
 173. The method of claim 172,wherein the 5′ overhang is 1-200 base pairs.
 174. The method of claim172, wherein the 3′ overhang is 1-100 base pairs.
 175. The method ofclaim 169, wherein any or all of the polynucleotide sequence encodingthe CRISPR enzyme, the first and the second guide sequence, the firstand the second tracr mate sequence or the first and the second tracrsequence, is/are RNA.
 176. The method of claim 169, wherein thepolynucleotides comprising the sequence encoding the CRISPR enzyme, thefirst and the second guide sequence, the first and the second tracr matesequence or the first and the second tracr sequence, is/are RNA and aredelivered via nanoparticles, exosomes, microvesicles, or a gene-gun.177. The method of claim 169, wherein the first and second tracr matesequence share 100% identity.
 178. The method of claim 169, wherein thefirst and second tracr sequence share 100% identity.
 179. The method ofclaim 169, wherein the CRISPR enzyme is a Cas9 enzyme.
 180. The methodof claim 179, wherein the CRISPR enzyme is SpCas9.
 181. The method ofclaim 180, wherein the CRISPR enzyme comprises one or more mutations ina catalytic domain.
 182. The method of claim 181, wherein the one ormore mutations is in a RuvC domain.
 183. The method of claim 182,wherein the one or more mutations is selected from the group consistingof D10A, E762A and D986A.
 184. The method of claim 183, wherein theCRISPR enzyme has the D10A mutation.
 185. The method of claim 181,wherein the one or more mutations is in a HNH domain.
 186. The method ofclaim 185, wherein the one or more mutations is selected from the groupconsisting of H840A, N854A and N863A.
 187. The method of claim 186,wherein the CRISPR enzyme has the H840A mutation.
 188. A method ofmodifying a DNA duplex at a locus of interest in a cell, the methodcomprising delivering to the cell: I. a first polynucleotide comprising:(a) a first guide sequence capable of hybridizing to a first targetsequence, (b) a first tracr mate sequence, and (c) a first tracrsequence; II. a second polynucleotide comprising: (a) a second guidesequence capable of hybridizing to a second target sequence, (b) asecond tracr mate sequence, and (c) a second tracr sequence; and III. athird polynucleotide comprising a sequence encoding a CRISPR enzyme andone or more nuclear localization sequences wherein (a), (b) and (c) insaid first and second polynucleotides are arranged in a 5′ to 3′orientation; wherein the first target sequence is on a first strand ofthe DNA duplex and the second target sequence is on the opposite strandof the DNA duplex, and when the first and second guide sequences arehybridized to said target sequences in the duplex, the 5′ ends of thefirst polynucleotide and the second polynucleotide are offset relativeto each other by at least one base pair of the duplex; wherein whentranscribed, the first and the second tracr mate sequences hybridize tothe first and second tracr sequences, respectively, and the first andthe second guide sequences direct sequence-specific binding of a firstand a second CRISPR complex to the first and second target sequencesrespectively, wherein the first CRISPR complex comprises the CRISPRenzyme complexed with (1) the first guide sequence that is hybridizableto the first target sequence, and (2) the first tracr mate sequence thatis hybridized to the first tracr sequence, wherein the second CRISPRcomplex comprises the CRISPR enzyme complexed with (1) the second guidesequence that is hybridizable to the second target sequence, and (2) thesecond tracr mate sequence that is hybridized to the second tracrsequence, and wherein said first strand of the DNA duplex is cleavednear said first target sequence, and said opposite strand of the DNAduplex is cleaved near said second target sequence, resulting in adouble strand break with a 5′ overhang or a 3′ overhang.
 189. A methodof modifying a DNA duplex at a locus of interest in a cell, the methodcomprising delivering to the cell a vector system comprising one or morevectors comprising: I. a first polynucleotide sequence comprising aregulatory element operably linked to (a) a first guide sequence capableof hybridizing to a first target sequence, and (b) at least one or moretracr mate sequences, II. a second polynucleotide sequence comprising asecond regulatory element operably linked to (a) a second guide sequencecapable of hybridizing to a second target sequence, and (b) at least oneor more tracr mate sequences, III. a third polynucleotide sequencecomprising a third regulatory element operably linked to a sequenceencoding a CRISPR enzyme, and IV. a fourth polynucleotide sequencecomprising a fourth regulatory element operably linked to a tracrsequence, wherein components I, II, III and IV are located on the sameor different vectors of the system wherein the first target sequence ison a first strand of the DNA duplex and the second target sequence is onthe opposite strand of the DNA duplex, and when the first and secondguide sequences are hybridized to said target sequences in the duplex,the 5′ ends of the first polynucleotide and the second polynucleotideare offset relative to each other by at least one base pair of theduplex; wherein when transcribed, the first and the second tracr matesequences hybridize to a tracr sequence, and the first and the secondguide sequences direct sequence-specific binding of a first and a secondCRISPR complex to the first and second target sequences respectively,wherein the first CRISPR complex comprises the CRISPR enzyme complexedwith (1) the first guide sequence that is hybridizable to the firsttarget sequence, and (2) the first tracr mate sequence that ishybridized to a tracr sequence, wherein the second CRISPR complexcomprises the CRISPR enzyme complexed with (1) the second guide sequencethat is hybridizable to the second target sequence, and (2) the secondtracr mate sequence that is hybridized to a tracr sequence, and whereinsaid first strand of the DNA duplex is cleaved near said first targetsequence, and said opposite strand of the DNA duplex is cleaved nearsaid second target sequence, resulting in a double strand break with a5′ overhang or a 3′ overhang.
 190. The method of claim 188 or 189,wherein said CRISPR enzyme is a nickase enzyme, optionally a Cas9 enzymecomprising at least one mutation in a catalytic domain.
 191. The methodof claim 190 wherein said at least one mutation is in the RuvC domainand optionally is selected from the group consisting of D10A, E762A andD986A, or is in the HNH domain and optionally is selected from the groupconsisting of H840A, N854A and N863A.
 192. The method according to anyone of claims 190 to 191, wherein said offset between the 5′ ends of thefirst polynucleotide and the second polynucleotide is greater than −8 bpor −278 to +58 bp or −200 to +200 bp or up to or over 100 bp or −4 to 20bp or +23 bp or +16 or +20 or +16 to +20 bp or −3 to +18 bp.
 193. Themethod according to any one of claims 190 to 192, wherein said cleavageof said first strand and of said opposite strand of the DNA duplexoccurs 3′ to a PAM (Protospacer adjacent motif) on each strand, andwherein said PAM on said first strand is separated from said PAM on saidopposite strand by from 30 to 150 base pairs.
 194. The method accordingto any one of claims 190 to 193, wherein said overhang is at most 200bases, at most 100 bases, or at most 50 bases.
 195. The method accordingto claim 194, wherein the overhang is at least 1 base, at least 10bases, at least 15 bases, at least 26 bases or at least 30 bases. 196.The method according to claim 194, wherein the overhang is between 34and 50 bases or between 1 and 34 bases.
 197. The method according to anyone of claims 188 to 197, wherein any or all of the polynucleotidesequence encoding the CRISPR enzyme, the first and the second guidesequence, the first and the second tracr mate sequence or the first andthe second tracr sequence, is/are RNA, and optionally wherein any or allof I, II and III are delivered via nanoparticles, exosomes,microvesicles, or a gene-gun.
 198. The method according to any one ofclaims 188 to 198, wherein the first and second tracr mate sequenceshare 100% identity and/or the first and second tracr sequence share100% identity.
 199. The method according to claim 188, wherein each ofI, II and III is provided in a vector, optionally wherein each isprovided in the same or a different vector.
 200. The method according toany one of claims 188 to 200, wherein said locus of interest comprises agene and wherein said method results in a change in the expression ofsaid gene, or in a change in the activity or function of the geneproduct.
 201. The method according to claim 201, wherein said geneproduct is a protein, and/or wherein said change in expression, activityor function is a reduction in said expression, activity or function.202. The method according to any one of claims 188 to 201, furthercomprising: delivering to the cell a double-strandedoligodeoxynucleotide (dsODN) comprising overhangs complimentary to theoverhangs created by said double strand break, wherein said dsODN isintegrated into the locus of interest; or delivering to the cell asingle-stranded oligodeoxynucleotide (ssODN), wherein said ssODN acts asa template for homology directed repair of said double strand break.203. The method according to any one of claims 188 to 201, which is forthe prevention or treatment of disease in an individual, optionallywherein said disease is caused by a defect in said locus of interest.204. The method according to claim 203, wherein the method is conductedin vivo in the individual or ex vivo on a cell taken from theindividual, optionally wherein said cell is returned to the individual.205. A kit or composition comprising: I. a first polynucleotidecomprising: (a) a first guide sequence capable of hybridizing to a firsttarget sequence, (b) a first tracr mate sequence, and (c) a first tracrsequence; II. a second polynucleotide comprising: (a) a second guidesequence capable of hybridizing to a second target sequence, (b) asecond tracr mate sequence, and (c) a second tracr sequence; and III. athird polynucleotide comprising a sequence encoding a CRISPR enzyme andone or more nuclear localization sequences wherein (a), (b) and (c) insaid first and second polynucleotides are arranged in a 5′ to 3′orientation; wherein the first target sequence is on a first strand of aDNA duplex and the second target sequence is on the opposite strand ofthe DNA duplex, and when the first and second guide sequences arehybridized to said target sequences in the duplex, the 5′ ends of thefirst polynucleotide and the second polynucleotide are offset relativeto each other by at least one base pair of the duplex, and optionallywherein each of I, II and III is provided in the same or a differentvector.
 206. Use of a kit or composition according to claim 205 in amethod according to any one of claims 188 to
 204. 207. Use of a kit orcomposition according to claim 205 in the manufacture of a medicament,optionally wherein said medicament is for the prevention or treatment ofa disease caused by a defect in said locus of interest.